CN101675069A - Process for the production of fine chemicals - Google Patents

Abstract

The present invention relates to a process for the production of the fine chemical in a microorganism, a plant cell, a plant, a plant tissue or in one or more parts thereof. The invention furthermorerelates to nucleic acid molecules, polypeptides, nucleic acid constructs, vectors, antisense molecules, antibodies, host cells, plant tissue, propagation material, harvested material, plants, microorganisms as well as agricultural compositions and to their use.

Description

Produce the method for fine chemicals

[0001.0.0.0] the present invention relates to produce the method for fine chemicals in microorganism, vegetable cell, plant, plant tissue or its one or more parts.The invention still further relates to material, plant, microorganism and Pestcidal compositions of nucleic acid molecule, polypeptide, nucleic acid construct, carrier, antisense molecule, antibody, host cell, plant tissue, reproductive material, results and uses thereof.

The application is based on the european patent application of previous proposition and require the right of these european patent applications: the European Patent Application No. 04105535.1 that on August 27th, 2004 proposed, the European Patent Application No. 04016615.9 that on July 15th, 2004 proposed, the European Patent Application No. 04015608.5 that on July 2nd, 2004 proposed, the European Patent Application No. 04018543.1 that on August 5th, 2004 proposed, the European Patent Application No. 04105689.6 that on August 23rd, 2004 proposed, the European Patent Application No. 04026008.5 that on November 3rd, 2004 proposed, the European Patent Application No. 04026007.7 that on November 3rd, 2004 proposed, the European Patent Application No. 04026057.2 that on November 4th, 2004 proposed, the European Patent Application No. 04026056.4 that on November 4th, 2004 proposed, the European Patent Application No. 04028670.0 that on December 3rd, 2004 proposed, the European Patent Application No. 04028671.8 that on December 3rd, 2004 proposed, the European Patent Application No. 04030101.2 that on December 18th, 2004 proposed, the European Patent Application No. 04106931.1 that on December 17th, 2005 proposed, the European Patent Application No. 04030391.9 that on December 22nd, 2004 proposed, the European Patent Application No. 05100166.7 that on January 10th, 2005 proposed, the European Patent Application No. 05103449.4 that on April 22nd, 2005 proposed, the European Patent Application No. 04107024.4 that on December 23rd, 2004 proposed, the European Patent Application No. 04030100.4 that on December 18th, 2004 proposed, the European Patent Application No. 05101970.1 that on March 14th, 2005 proposed, the European Patent Application No. 04107025.1 that on December 28th, 2004 proposed, the European Patent Application No. 05104781.9 that on May 27th, 2005 proposed, the European Patent Application No. 05100704.5 that on January 26th, 2005 proposed, the European Patent Application No. 05103283.7 that on April 26th, 2005 proposed, the European Patent Application No. 05103455.1 that on April 22nd, 2005 proposed, the European Patent Application No. 05103164.9 that on April 20th, 2005 proposed, the European Patent Application No. 05103428.8 that on April 27th, 2005 proposed, the European Patent Application No. 05104479.0 that on May 25th, 2005 proposed, the European Patent Application No. 05104496.4 that on May 25th, 2005 proposed, the European Patent Application No. 05105001.1 that on June 6th, 2005 proposed, the European Patent Application No. 05104874.2 that on June 3rd, 2005 proposed, the European Patent Application No. 05105345.2 that on June 10th, 2005 proposed, the European Patent Application No. 05104630.8 that on May 30th, 2005 proposed, the European Patent Application No. 05104761.1 that on June 1st, 2005 proposed, the European Patent Application No. 05104811.4 that on June 2nd, 2005 proposed, the European Patent Application No. 05104818.9 that on June 2nd, 2005 proposed, the European Patent Application No. 05105021.9 that on June 8th, 2005 proposed, the European Patent Application No. 05105028.4 that on June 8th, 2005 proposed, the European Patent Application No. 05105136.5 that on June 13rd, 2005 proposed, the European Patent Application No. (Customs Assigned Number NAE419/05EP) that on May 27th, 2005 proposed, the European Patent Application No. 05105508.5 that on June 21st, 2005 proposed, the European Patent Application No. 05105575.4 that on June 22nd, 2005 proposed, the European Patent Application No. 05105510.1 that on June 21st, 2005 proposed, the European Patent Application No. 05105401.3 that on June 17th, 2005 proposed, the European Patent Application No. 05105405.4 that on June 17th, 2005 proposed, the European Patent Application No. (Customs Assigned Number NAE42505EP) that on May 27th, 2005 proposed, the European Patent Application No. 05105570.5 that on June 22nd, 2005 proposed, the European Patent Application No. 05105406.2 that on June 17th, 2005 proposed, the European Patent Application No. 05105624.0 that on June 23rd, 2005 proposed, the European Patent Application No. 05105571.3 that the European Patent Application No. 05105643.0 that on June 23rd, 2005 proposed and on June 22nd, 2005 propose.The full content of above referenced patent application is quoted as a reference in the text.

[0002.0.0.0] amino acid is used for many kind industry, comprises food, animal-feed, makeup, pharmacy and chemical industry.Amino acid such as D, L-methionine(Met), L-Methionin or L-Threonine are used for animal-feed industry.Indispensable amino acid: Xie Ansuan, leucine, Isoleucine, Methionin, Threonine, methionine(Met), tyrosine, phenylalanine and tryptophane are to the nutrition particularly important of human and many domestic animal species.Glycine, L-methionine(Met) and tryptophane all are used for pharmaceutical industry.Glutamine, Xie Ansuan, leucine, Isoleucine, Histidine, arginine, proline(Pro), Serine and L-Ala are used for pharmacy and cosmetic industry.Threonine, tryptophane and D, L-methionine(Met) are widely used fodder additives (Leuchtenberger, W. (1996) Amino acids-technicalproduction and use, Rehm etc. (volume) Biotechnology the 6th volume, the 14a chapter, the 466-502 page or leaf among the VCHWeinheim).And, amino acid is used for synthesizing amino acid and protein as the precursor that is suitable for chemical industry, as N-acetylcystein, S-carboxymethyl-L-halfcystine, (S)-5-hydroxyryptophan and Ullmann ' s Encyclopedia of Industrial Chemistry, the A2 volume, the 57-97 page or leaf, VCH Weinheim, other materials described in 1985 synthetic.

[0003.0.0.0] annual now generation surpasses 1,000,000 tons of amino acid; Its marketable value totals over 2,500,000,000 dollars.They produce by the method for four kinds of competitions at present: extract from protein hydrolystate, for example L-Gelucystine, L-leucine or L-tyrosine; Chemosynthesis, for example D, L-methionine(Met); In enzyme or cell reactor, transform, for example the L-phenylalanine from precursor; Produce with the fermentation of technical scale culturing bacterium with passing through, described bacterium has been developed a large amount of required molecules of interest of generation justacrine.The biology that is specially adapted to this purpose is the Corynebacterium glutamicum (Corynebacterium glutamicum) that is used to produce L-Methionin for example or L-L-glutamic acid.Other are for example L-Threonine, L-tryptophane, L-aspartic acid and L-phenylalanine by the amino acid that fermentation produces.

The biosynthesizing of [0004.0.0.0] natural amino acid in the biology that can produce them (as bacterium) fully characterized, bacterium amino acid bio summary synthetic and that regulate can be consulted Umbarger, H.E. (1978) Ann.Rev.Biochem.47:533-606.

[0005.0.0.0] is known to the particularly Corynebacterium glutamicum generation amino acid of coryneform bacterial strains that ferments.Because they are extremely important, so its production method is always in continuous improvement.The improvement of method can relate to the measure about the fermentation technique aspect, for example stirs and the oxygen supply; Or about the measure of nutritional medium composition technical elements, the sugared concentration when for example fermenting; Or relate to the inspection of product, for example pass through ion-exchange chromatography; Or relate to the internal performance characteristic of microorganism itself.Also be used to produce amino acid from other bacteriums that belong to as intestinal bacteria or genus bacillus.Researched and developed the mass mutation bacterial strain by bacterial strain screening, they produce a class required compound from the sulfur-bearing fine chemicals.With regard to the generation of specific molecular, select to improve the performance of described microorganism by mutagenesis, selection and mutant.Also developed the method that produces methionine(Met).Obtain by this way for example metabolic antagonist (as analogue Alpha-Methyl methionine(Met), ethionine, nor-leucine, N-ethanoyl nor-leucine, S-trifluoromethyl homocysteine, 2-amino-5-heprenoitic acid, selenomethionine, methionine sulfoxide amine, methoxine, the 1-aminocyclopentanecarboxylic acid of methionine(Met)) being had resistance or being metabolite defective type with adjusting importance and the bacterial strain that produces sulfur-bearing fine chemicals (as the L-methionine(Met)).Yet, these shortcomings that develop the method that produces methionine(Met) for its output is too low can not economic exploitation, so they still can not be competed with chemosynthesis now.

[0006.0.0.0] Zeh (2001:792-802) described and suppress threonine synthase by known antisense technology and improve methionine(Met) content in the potato plants by Plant Physiol., 127 volumes.This causes active reduction of threonine synthase and threonine content in the plant does not reduce.This technology is very complicated, and enzymic activity must suppress in the mode of highly distinguishing, otherwise the amino acid nutrient defective will take place, and plant will stop growing.

[0007.0.0.0] US 5,589, thus 616 taught by in dicotyledons, crossing and expressed monocotyledonous storage protein produces a greater number in plant amino acid.WO 96/38574, WO97/07665, WO 97/28247, US 4,886,878, US 5,082,993 and US 5,670,635 have followed this method.This means in whole aforementioned intellecture properties, in plant, expressed different protein or polypeptide.Described protein or polypeptide should play a role as the amino acid pond.The additive method that improves amino acid (as Methionin) is set forth in WO 95/15392, WO 96/38574, WO89/11789 or WO 93/19190.In this case, the specific enzymes in the amino acid biosynthetic pathway (as the dihydrodipicolinate synthase) is imbalance.This causes the raising of lysine production in the different plants.The another kind of method that improves amino acid levels in the plant is disclosed in EP-A-0271408.EP-A-0271408 has taught the mutagenesis plant and has selected with the inhibitor of some enzyme in the amino acid biosynthetic pathway subsequently.

[0008.0.0.0] is used for improve generation L-amino acid whose coryneform bacterial strains to the influence that amino acid produces with the recombinant DNA technology method by increase single amino acids biosynthesis gene and examination over several years.

[0009.0.0.0] as previously mentioned, indispensable amino acid is essential to human and a lot of Mammalss as domestic animal.The L-methionine(Met) as the methyl group donor to the biosynthesizing of for example choline, creatine, suprarenin, base and RNA and DNA, Histidine, transmethylation after forming S-adenosylmethionine or all extremely important to the formation of halfcystine as the sulfydryl donor.In addition, the L-methionine(Met) shows the active effect to dysthymia disorders.

The quality that [0010.0.0.0] improves grain and animal-feed is the vital task of food and fodder industry.This is necessary, because for example some amino acid that exists in the plant is very limited for mammiferous demand.Because other amino acid become limiting factor suddenly, the amino acid that substantially exceeds specific concentrations in the food does not have other active effects to the utilization of food, and therefore particularly advantageous for the quality of grain and animal-feed is equilibrated aminogram as far as possible.Can only further improve quality by adding other amino acid that become limiting factor under these conditions.Target adds the limited amino acid of sintetics form must extremely careful carrying out, unbalance to avoid amino acid.For example, add the digestion of indispensable amino acid stimulating protein, especially may cause second kind or the insufficient situation of the third limited amino acid.In feeding experiment (for example casein is fed experiment), additionally provide methionine(Met) limited in the casein to show steatosis in the liver, described steatosis can only alleviate by extra interpolation tryptophane.

[0011.0.0.0] therefore for guaranteeing the high-quality of food and animal-feed, is necessary to add multiple amino acids so that be fit to biological with balance mode.

[0012.0.0.0] therefore the objective of the invention is to research and develop inexpensive L-methionine(Met) biosynthetic means.The L-methionine(Met) becomes the highest limiting amino acid of the frequency of occurrences with the Methionin or the Threonine (depending on organism) of one of two amino acid.

[0013.0.0.0] finds now, realized this purpose by the embodiment that is characterized in the inventive method as herein described and claims is provided.

[0014.0.0.0] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and fine chemicals is a methionine(Met) thus, therefore, in the present invention, term " fine chemicals " such as in the literary composition use relate to " methadone ".In addition, in another embodiment, term " fine chemicals " such as in the literary composition use also relate to the fine chemicals composition that comprises methionine(Met).

[0015.0.0.0] in one embodiment, term " fine chemicals " or " each fine chemicals " are meant the L-methionine(Met).Term in the entire description " fine chemicals " or " each fine chemicals " are meant methionine(Met), preferred L-methionine(Met), the salt of its free form, ester or acid amides or be bonded to proteinic methionine(Met), preferably L-methionine(Met).In preferred embodiments, term " fine chemicals " is meant L-methionine(Met) or its salt of free form or is bonded to proteinic L-methionine(Met).In one embodiment, term " fine chemicals " and term " each fine chemicals " are meant at least a active chemical compound of above-mentioned fine chemicals that has.

[0016.0.0.0] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, increase or produce one or more YLR375W, YBL015w, YER173w, YOR084w and/or b1829 and/or b4232, b0464, b1343, b2414 and/or b2762 activity of proteins, perhaps have by Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in nucleic acid molecule encoded polypeptide sequence activity of proteins and

(b) in allowing described biology, produce under the condition that fine chemicals is methionine(Met) or the fine chemicals that comprises methionine(Met) and make biological growth.

Therefore, the present invention relates to may further comprise the steps the method that produces fine chemicals:

(a) in non-human being or its one or more parts, increase or produce have Table II the 3rd row, 1-5 is capable and/or 334-338 capable shown in one or more activity of proteins of activity of proteins, perhaps have by Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in nucleic acid molecule encoded polypeptide sequence one or more activity of proteins and

(b) under the condition that allows generation fine chemicals, particularly methionine(Met), make biological growth.

[0016.1.0.0]./.

" containing/comprise " of using in [0017.0.0.0] this specification sheets and grammatical variants thereof are used for explanation and have described feature, integral body, step or component or its group, but do not get rid of existence or add one or more other features, integral body, step, component or its group.The term that uses in this specification sheets " Table I " is used for the content of instruction card I A and Table I B.The term that uses in this specification sheets " Table II " is used for illustrating the content of Table II A and Table II B.The term that uses in this specification sheets " Table I A " is used for the content of instruction card I A.The term that uses in this specification sheets " Table I B " is used for the content of instruction card I B.The term that uses in this specification sheets " Table II A " is used to illustrate the content of Table II A.The term that uses in this specification sheets " Table II B " is used to illustrate the content of Table II B.In a preferred embodiment, term " Table I " is meant Table I B.In a preferred embodiment, term " Table II " is meant Table II B.

[0018.0.0.0] preferably, this method further comprises the step that reclaims biosynthetic fine chemicals from substratum biological and/or that be used for cultivating or keep described biology.Term " recovery " refers to different purity separate fine chemical, this refers to gather in the crops the biomaterial that contains this fine chemicals on the one hand and is not further purified, the purity that refers to fine chemicals on the other hand is between 5% and 100%, and preferred purity is in 10% and 99% scope.In one embodiment, purity is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[0019.0.0.0] advantageously, the method that produces this fine chemicals causes that this fine chemicals produces and strengthens.Term " enhanced " or " raising " refer to and compare as the reference of giving a definition, the generation of fine chemicals improves 10% at least, 20%, 30%, 40%, or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or 500%, described for example relatively being meant compared with the biophase that activity of proteins is not carried out described modification, described protein has Table II the 3rd row, activity of proteins shown in the capable and/or 334-338 of 1-5 is capable perhaps has by Table I the 5th or 7 row, the coded activity of proteins of nucleic acid molecule shown in the capable and/or 334-338 of 1-5 is capable.

[0020.0.0.0] finds surprisingly, in Arabidopis thaliana (Arabidopsis thaliana) at least a Table II of transgene expression the 3rd row, 1-4 capable shown in yeast saccharomyces cerevisiae (Saccharomycescerevisiae) protein, and/or at least a Table II the 3rd row, the 5th row and/or 334-338 capable shown in e. coli k12 (Escherichia coli K12) protein, the methionine(Met) content that transforms plant is increased.

[0021.0.0.0] according to the present invention, term " biology " all is interpreted as relating to the non-human being in this article, is specifically related to animal or plant or microorganism.In addition, term " animal " all is interpreted as relating to the non-human animal in this article.According to the present invention, the technician knows that anionic compound such as acid are present in the aqueous solution with the balance between acid and its salt according to the pK value of pH value in each chamber in cell or the organism and acid.Depend on the intensity (pK) and the pH of acid, salt or free acid are main.Therefore, term " fine chemicals ", term " each fine chemicals " or term " acid " or mention in the title used during with anionic compound with according to its existing in the anionic form and the neutral condition of compound of aqueous environment relevant.

The sequence of [0022.0.0.0] yeast saccharomyces cerevisiae YLR375w has been published in Johnston, Nature387 (6632 supplementary issue), 87-90,1997, and Goffeau, Science 274 (5287), 546-547,1996, and its activity " relate to the absorption of preceding-tRNA montage and branched-chain amino acid; YLR375wp ".Therefore, in one embodiment, method of the present invention comprise as shown in literary composition from the gene product of the relating to of yeast saccharomyces cerevisiae " preceding-tRNA montage and branched-chain amino acid are taken in " or the purposes of its homologue, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of yeast saccharomyces cerevisiae YBL015w has been published in Goffeau, and Science 274 (5287), 546-547,1996 and Feldmann, EMBO J., 13,5795-5809, in 1994, and its activity be defined as " contain seminose glycoprotein in conjunction with concanavalin A; Ach1p ".In another piece reference, activity is described as " Acetyl-CoA hydrolase ".Therefore, in one embodiment, method of the present invention comprises " contain seminose glycoprotein in conjunction with concanavalin A from yeast saccharomyces cerevisiae as shown in literary composition; Ach1p " or the purposes of its homologue, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).Therefore, in one embodiment, method of the present invention comprises " Acetyl-CoA hydrolase " or the purposes of its homologue from yeast saccharomyces cerevisiae as shown in literary composition, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of yeast saccharomyces cerevisiae YER173w has been published in Dietrich, Nature 387 (6632 supplementary issue), 78-81,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity be defined as " check position protein participates in the activation of dna damage and reduction division pachytene stage check position; Rad17p-Mec3p-Dc1p is loaded into the subunit of the clip loading bin (clamp loader) on the DNA, the proteinic homologue of people and schizosaccharomyces pombe (S.pombe) Rad17; Rad24p ".Therefore, in one embodiment, method of the present invention comprises " Rad17p-Mec3p-Dc1p is loaded into the subunit of the clip loading bin on the DNA " or the purposes of Rad24p or Rad17 protein or its homologue from " the check position protein; participate in the activation of dna damage and reduction division pachytene stage check position " of yeast saccharomyces cerevisiae or its as shown in literary composition, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of yeast saccharomyces cerevisiae YOR084w has been published in Dujon, Nature 387 (6632 supplementary issue), and 98-102,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity is defined as the peroxysome matrix lipase of supposition.Therefore, in one embodiment, method of the present invention comprises being the purposes of thing from the peroxysome matrix lipase of the supposition of yeast saccharomyces cerevisiae or with it as shown in literary composition, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of the b1829 of e. coli k12 has been published in Blattner, Science277 (5331), and 1453-1474,1997, and its activity is defined as heat shock protein.Therefore, in one embodiment, method of the present invention comprises the purposes from colibacillary " heat shock protein " or its homologue as shown here, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).In one embodiment, strengthen in the methods of the invention or for example produced activity from colibacillary htpX heat shock protein or its homologue.For example the homologue of htpX heat shock protein also is interpreted as having protease activity.Therefore, in one embodiment, strengthen proteolytic enzyme, preferred heat shock protein(HSP) enzyme in the methods of the invention, be more preferably the activity of htpX proteolytic enzyme or its homologue, to produce fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of the b0464 of e. coli k12 has been published in Blattner, Science277 (5331), and 1453-1474,1997, and its activity is defined as " the transcription repression albumen of multiple medicines efflux pump (TetR/AcrR family) ".Therefore, in one embodiment, method of the present invention comprises the purposes from colibacillary " the transcription repression albumen of multiple medicines efflux pump (TetR/AcrR family) " or its homologue as shown in literary composition, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).In one embodiment, in the methods of the invention, increase or for example produced, be preferably and have control of transcribing and/or dna binding activity from colibacillary " possible transcription repression albumen mtrr " protein of superfamily or the activity of its homologue.Therefore, in one embodiment, strengthened the activity of " the transcription repression albumen of multiple medicines efflux pump (TetR/AcrR family) " or its homologue in the method for the invention, to produce fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of the b1343 of e. coli k12 has been published in Blattner, Science277 (5331), and 1453-1474,1997, and its activity is defined as the ATP dependenc RNA helicase that is stimulated by 23S rRNA.Therefore, in one embodiment, method of the present invention comprises the purposes from colibacillary " by the ATP dependenc RNA helicase of 23S rRNA stimulation " or its homologue as shown in literary composition, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).In one embodiment, increase in the method for the invention or for example produced and in rRNA processing or translation, to have an active activity of proteins from colibacillary protein or its homologue.Therefore, in one embodiment, having strengthened in the method for the invention by the ATP dependenc RNA helicase of 23S rRNA stimulation or the activity of its homologue, is methionine(Met) to produce fine chemicals in biological or its part, particularly increases the quantity of free or combining form methionine(Met).

The sequence of the b2414 of e. coli k12 has been published in Blattner, Science277 (5331), and 1453-1474,1997, and its activity is defined as PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit.Therefore, in one embodiment, method of the present invention comprises the intestinal bacteria " PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit " as shown in literary composition or the purposes of its homologue, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).In one embodiment, increase or produced the activity of " threonine dehydra(ta)se " superfamily protein in the intestinal bacteria or its homologue in the method for the invention, preferably have at amino acid bio synthetic, halfcystine-aromatic series biosynthesizing, halfcystine-die aromatischen Aminosaeuren degraded, the nitrogen and the sulphur of aspartate family utilize biosynthesizing, aspartic acid family amino acid degradation, the biosynthesizing of sulfuric acid and L-cysteine derivative, biosynthesizing from the secondary species of primary amino acid, from glycine, the biosynthesizing of L-Serine and L-L-Ala deutero-secondary species, pyridoxal phosphate bonded activity more preferably has " PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit " activity.Therefore, in one embodiment, strengthened the activity of " PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit " or its homologue in the method for the invention, to produce fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of the b2762 of e. coli k12 has been published in Blattner, Science277 (5331), and 1453-1474,1997, and its activity is defined as 3 '-adenosine phosphate 5 '-phosphinylidyne sulfuric acid (PAPS) reductase enzyme.Therefore, in one embodiment, method of the present invention comprises the purposes from colibacillary " 3 '-adenosine phosphate 5 '-phosphinylidyne sulfuric acid (PAPS) reductase enzyme " or its homologue as shown in literary composition, it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).In one embodiment, strengthen in the method for the invention or for example produced activity from colibacillary " 3 '-adenosine phosphate 5 '-phosphinylidyne sulfuric acid (PAPS) reductase enzyme " superfamily protein or its homologue, preferably have activity in the utilization of halfcystine biosynthesizing, nitrogen and sulphur, amino acid bio are synthetic, what be more preferably has " 3 '-adenosine phosphate 5 '-phosphinylidyne sulfuric acid (PAPS) reductase enzyme " activity.Therefore, in one embodiment, strengthened the activity of " 3 '-adenosine phosphate 5 '-phosphinylidyne sulfuric acid (PAPS) reductase enzyme " or its homologue in the method for the invention, to produce fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The sequence of the b4232 of e. coli k12 has been published in Blattner, Science277 (5331), and 1453-1474,1997, and its activity is defined as fructose-1.Therefore, method of the present invention comprises the purposes from colibacillary " fructose-1; 6-diphosphatase " or its homologue as shown in literary composition, and it is used for producing fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).In one embodiment, strengthen in the method for the invention or for example produced activity from colibacillary " fructose diphosphatase " superfamily protein or its homologue, preferably in C-compound and carbohydrate metabolism, C-compound and carbohydrate utilization, energy, glycolysis-and gluconeogenesis, plastid, photosynthesis, has activity, what be more preferably has " fructose-1 " activity.Therefore, in one embodiment, strengthened the activity of " fructose-1 " or its homologue in the method for the invention, to produce fine chemicals in biological or its part is methionine(Met), particularly increases the quantity of free or combining form methionine(Met).

The homologous compound (homologue) of [0023.0.0.0] gene product of the present invention (=homologue (homolog)) can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with at least a Table I the 3rd row, 1-5 is capable and/or 334-338 capable shown in protein, for example have by contain Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in Table II 1-4 is capable in the polypeptide homologue of any one be to have a same or similar active homologue, particularly active increasing causes that fine chemicals content increases in the biology, and described homologue is from eukaryote.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 5th row and/or 334-338 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 1-4 are capable is to have a same or similar active homologue, and particularly active increasing causes that fine chemicals content increases in biology or its part, and described homologue is from fungi.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 5th row and/or 334-338 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from Proteobacteria (Proteobacteria).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 1-4 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from Ascomycota (Ascomycota).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 5th row and/or 334-338 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from γ-distortion Gammaproteobacteria (Gammaproteobacteria).In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, the 1-4 row, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from yeast (Saccharomycotina).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 5th row and/or 334-338 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from enterobacteria order (Enterobacteriales).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 1-4 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from yeast guiding principle (Saccharomycetes).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 5th row and/or 334-338 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from enterobacteriaceae (Enterobacteriaceae).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 1-4 were capable, particularly active increasing causes fine chemicals content increase in the biology, and described homologue is from Saccharomycetes (Saccharomycetales).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 5th row and/or 334-338 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from Escherichia (Escherichia).In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 1-4 were capable, particularly active increasing causes fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetaceae (Saccharomycetaceae).In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 1-4 are capable is to have a same or similar active homologue, and particularly active increasing causes that fine chemicals content increases in biology or its part, and described homologue is from the yeast guiding principle.

The homologue of polypeptide shown in [0023.1.0.0] Table II the 3rd row, 1-4 are capable can be by Table I the 7th row, 1-4 capable shown in the polypeptide of nucleic acid molecule encoding, perhaps can be Table II the 7th row, 1-4 capable shown in polypeptide.The homologue of polypeptide shown in Table II the 3rd row, the 5th row or 334-338 are capable can be by Table I the 7th row, the 5th row or 334-338 capable shown in the polypeptide of nucleic acid molecule encoding, perhaps can be Table II the 7th row, the 5th row and/or 334-338 capable shown in polypeptide.

In the literary composition other homologue has been described below [0024.0.0.0].

[0025.0.0.0] is according to the present invention, methionine(Met), preferred L-methionine level increase in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", perhaps have be used for that protein of the present invention for example has Table II the 3rd row, 1-5 is capable and/or 334-338 capable shown in the activity of proteins of protein active.In preferred embodiments, protein or polypeptide have Table II the 3rd row, 1-5 is capable and/or 334-338 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, any one proteinic biology or the enzyme activity shown in the capable and/or 334-338 of 1-5 is capable, if promptly with Table II the 3rd row, the protein of any one yeast saccharomyces cerevisiae shown in 1-4 is capable is compared and/or is listed as with Table II the 3rd, the protein of any one e. coli k12 was compared shown in the 5th row and/or 334-338 were capable, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.0] in one embodiment, if it comes comfortable the evolution to go up biology with the biological apart from each other of expressing it, polypeptide of the present invention or the polypeptide that is used for the inventive method are still given described activity, as increase fine chemicals in biological or its part.For example play eozoan and express biology from different sections, order, guiding principle or door.

[0025.2.0.0] in one embodiment, if polypeptide come comfortable evolve go up with biophase shown in Table I the 4th row apart from nearer biological and in evolution with in the biology of initial biological apart from each other, expressing, polypeptide of the present invention or the polypeptide that is used for the inventive method can be given described activity, as increase fine chemicals in biological or its part.For example just eozoan is biological from different sections, order, guiding principle or door with expression, and biology comes from identical section, order, guiding principle or door shown in first eozoan and Table I the 4th row.

[0026.0.0.0] term " raising ", " rising ", " extension ", " enhanced ", " improved " or " expansion " relate to corresponding character change in biology, biological part (as tissue, seed, root, leaf, flower etc.) or the cell, and are used interchangeably.Preferably, under following situation, if promptly improve or strengthen and relate to active raising of gene product or enhancing, and do not rely on the specific activity of the quantity of gene product or gene product or whether both improve or strengthen, when whether quantity, stability or the translation efficiency that does not also rely on the nucleotide sequence of encoding gene product or gene improves or strengthens, then the overall activity in the volume be improve or enhanced.Term " minimizing ", " reduction " or " disappearance " relate to corresponding character change in biology, biological part (as tissue, seed, root, leaf, flower etc.) or the cell.Preferably, under following situation, if promptly reduce, reduce or lack and relate to the active minimizing of gene product, reduction or disappearance, and do not rely on the specific activity of the quantity of gene product or gene product or whether both reduce, reduce or lack, when whether quantity, stability or the translation efficiency that does not also rely on the nucleotide sequence of encoding gene product or gene reduced, reduces or lack, then the overall activity in the volume was reduced, reduces or lacks.

[0027.0.0.0] term " raising " or " reduction " relate to corresponding character change in biological, biological partly (as tissue, seed, root, leaf, flower etc.) or cell.Preferably, relate to the active raising of gene product when improving or strengthening, and do not rely on the specific activity of the quantity of gene product or gene product or both and whether improve or produce, whether also do not rely on the nucleotide sequence of encoding gene product or quantity, stability or the translation efficiency of gene and improve, then the overall activity in the volume is enhanced.

[0028.0.0.0] is in " change of character ", be to be understood that, the content of the activity of gene product, expression level or quantity or metabolite is altered for contrast, reference or the wild-type of respective volume in the designated volume, comprises the from the beginning generation of activity or expression.

[0029.0.0.0] term " raising " or " reduction " comprise that described character only changes to some extent or regulates in the part of research object of the present invention, for example modify and be found in cellular compartment (as organoid), or in the plant part (as tissue, seed, root, leaf, flower etc.), if but detect whole research object (being complete cell or plant), then detect less than.Preferably, it is intracellular improving or reducing, so improves in term " active raising " or " raising of metabolite content " relates to wild-type cell is compared the cell.In any case, such as in the literary composition use, term improves or reduces variation or the adjusting that also is included in the character in the above-mentioned complete biology.

[0030.0.0.0] therefore, term " raising " or " reduction " are meant that the amount of specific activity, preferred compound or the metabolite (for example each fine chemicals of polypeptide, nucleic acid molecule or the present invention or coding mRNA or DNA) of enzyme can improve or reduce in volume.

[0031.0.0.0] term " wild-type ", " contrast " or " reference " are used interchangeably and can are the not modification of the inventive method described in the scripture or the cell of handling or biological part (as organoid or tissue) or biological, especially microorganism or plant.Therefore, will be as the cell of wild-type, contrast or reference or biological part (as organoid or tissue) or biological (especially microorganism or plant) as much as possible corresponding to cell, biology or its part, and any other character except that the inventive method result is identical as far as possible with research object of the present invention.Therefore, the processing of wild-type, contrast or reference is wanted identical or identical as far as possible, promptly have only the conditioned disjunction character that does not influence test character quality just can be different.

[0032.0.0.0] preferably anyly relatively will carry out under conditions of similarity.Term " conditions of similarity " is meant all conditions, and for example cultivation or growth conditions, condition determination (for example damping fluid composition, temperature, substrate, pathogenic strains, concentration etc.) keep identical between experiment to be compared.

[0033.0.0.0] " reference ", " contrast " or " wild-type " are not preferably and modify or handle and other any character object similar as far as possible to research object of the present invention according to the inventive method described herein, as organoid, cell, tissue, biology, especially plant or microorganism.With reference to, contrast or wild-type at genome, to transcribe group, protein group or metabolism prescription face similar as far as possible to research object of the present invention.Preferably, term " reference " " contrast " or " wild-type " organoid, cell, tissue or biology (especially plant or microorganism) relate to organoid of the present invention, cell, tissue or biology (especially plant or microorganism) or its part in heredity much at one, preferred 95%, more preferably 98% even more preferably 99.00%, particularly 99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99.999% or organoid, cell, tissue or the biology (especially plant or microorganism) of higher identity.Most preferred " reference ", " contrast " or " wild-type " are, except the gene product of giving reaction or active nucleic acid molecule or its coding is revised, operates, is exchanged or import by the method according to this invention, with identical object in biology, cell or the organoid heredity that the inventive method is used, as organoid, cell, tissue or biology.

[0034.0.0.0] preferably, with reference to, contrast or wild-type only at polypeptide of the present invention or be used for aspect the cytoactive of polypeptide of the inventive method different with research object of the present invention, these differences be since for example nucleic acid molecule level of the present invention improve, or due to polypeptide of the present invention or the specific activity that is used for the polypeptide of the inventive method improve.For example, its have Table II the 3rd row, 1-5 is capable and/or 334-338 capable shown in protein or by Table I the 5th row, 1-5 is capable and/or 334-338 capable shown in the protein of nucleic acid molecule encoding or its homologue (as Table I the 7th row, 1-5 is capable and/or 334-338 capable shown in homologue) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows the fine chemicals quantity that increases.

[0035.0.0.0] is when not providing only owing to not being the contrast that the inventive method object is different from object of the present invention, when reference or wild-type, then contrast, reference or wild-type can be such biologies, wherein reverse or closed the active regulation and control factor that fine chemicals increases or nucleic acid molecule as described herein is expressed of giving, this can realize by the expression that for example knocks out the genes involved product, for example passes through Antisense Suppression, the inactivation of activator or agonist, the activation of inhibitor or antagonist, suppress by adding inhibition antibody, add active compound (as hormone), introduce negative dominant mutant etc.For example can cause enzymic activity to suppress or unstable or suddenly change in conjunction with the deactivation point that the ability of cofactor is suppressed etc. and to knock out gene product by introducing.

[0036.0.0.0] therefore, preferred reference object is the origin object of the inventive method.Preferably behind total RNA, DNA or protein or activity or amount stdn of expressing and normalization method, compare reference and research object of the present invention to for example reference gene (as house-keeping genes such as ubiquitin, Actin muscle or ribosomal proteins).

There is a series of mechanism in [0037.0.0.0], can directly or indirectly influence output, production and/or the production efficiency of fine chemicals by the modification of these machine-made egg white matters (polypeptide for example of the present invention or be used for the polypeptide of the inventive method).

[0038.0.0.0] for example can improve the molecular amounts or the specific activity of polypeptide or nucleic acid molecule.If from the beginning polypeptide of the present invention or nucleic acid express, then can produce more substantial fine chemicals in the biology that lacks described protein active.Yet, by for example regulation and control of amplification gene quantity, modifying factor, or the stability of the corresponding gene product of the stability of raising corresponding mRNA or nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding, or introduce from other biological and by the homologous gene of differently adjusting (regulating as non-feedback is responsive), can improve the natural expression of gene that exists in the biology.

This also is applied to make up expression and other enzyme of each fine chemicals biosynthetic pathway or the expression of instrumentality (as useful enzyme or instrumentality in each fine chemicals is synthetic) that improves nucleic acid molecule of the present invention or its gene product similarly [0039.0.0.0].

[0040.0.0.0] raising of the present invention, reduction or regulation and control can be composing types, for example because stable permanent transgene expression, perhaps because the stable sudden change of the corresponding native gene of code book invention nucleic acid molecule or the inventive method nucleic acid molecule used therefor, perhaps because to giving polypeptide of the present invention or being used for the expression of gene of expression of polypeptides of the inventive method or the regulation and control of behavior; Also can be instantaneous, for example because instantaneous conversion or add instrumentality (as agonist or antagonist) temporarily; But or induction type, for example with carry be positioned at inducible promoter control down nucleic acid molecule of the present invention or the induced construct of the inventive method nucleic acid molecule used therefor transform and adding inductor (as tsiklomitsin or as mentioned below).

[0041.0.0.0] compares with contrast, reference or wild-type, the polypeptide active increase preferably at least 5% in cell, tissue, organoid, organ or biological or its part, preferably at least 20% or at least 50%, especially preferably at least 70%, 80%, 90% or more, especially especially preferably at least 200%, most preferably at least 500%.

The specific activity of coded polypeptide of [0042.0.0.0] nucleic acid molecule of the present invention or polypeptide of the present invention can as be shown in the examplesly be tested.Particularly, the detection that the expression of described protein in cell (as vegetable cell or microorganism) reaches each fine chemicals level raising compared with the control is test easily, and can be as enforcement as described in the current state of the art.

[0043.0.0.0] term " raising " comprise from the beginning compound or activity introduced cell or comprise before never detect compound or activity, in other words be " generation ".

[0044.0.0.0] therefore, hereinafter, term " raising " also comprises term " generation " or " stimulation ".Active raising self shows as the increase of fine chemicals.

[0045.0.0.0] in one embodiment, under the situation that yeast saccharomyces cerevisiae protein YLR375W or its homologue (shown in Table II the 5th or 7 row, the 1st row) activity is enhanced, preferably, give fine chemicals be increased in 110% and 300% or higher between.

In one embodiment, under the situation that yeast saccharomyces cerevisiae protein YBL015w or acetyl-CoA lytic enzyme or its homologue (shown in Table II the 5th or 7 row, the 2nd row) activity is enhanced, preferably, give fine chemicals be increased in 110% and 300% or higher between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173w or its homologue (for example shown in Table II the 5th or 7 row, the 3rd row), for example participate in dna damage and reduction division pachytene stage check position activatory check position protein; Rad17p-Mec3p-Ddc1p is loaded into subunit or Rad24p or its homologue of the clip loading bin of DNA, Rad17 as people or schizosaccharomyces pombe, activity situation about being enhanced under, preferably, give fine chemicals be increased in 110% and 200% or higher between.

In one embodiment, under the situation that the peroxysome matrix lipase of yeast saccharomyces cerevisiae protein YOR084w or supposition or its homologue (shown in Table II the 5th or 7 row, the 4th row) activity is enhanced, preferably, give fine chemicals be increased in 110% and 350% or higher between.

In one embodiment, be enhanced in e. coli k12 protein b1829 or its homologue (shown in Table II the 5th or 7 row, the 5th row) activity, for example protease activity is enhanced, preferably the heat shock protein activity is enhanced, more preferably under the situation that htpX protein or its homologue activity are enhanced, preferably, give fine chemicals be increased in 110% and 400% or higher between.

In one embodiment, (be listed as at e. coli k12 protein b4232 or its homologue as Table II the 5th or 7, shown in the 338th row) activity is enhanced, for example fructose diphosphatase superfamily protein active is enhanced, preferably participate in C-compound and carbohydrate metabolism, C-compound and carbohydrate utilization, energy, glycolysis-and gluconeogenesis, plastid and/or photosynthetic protein active are enhanced, fructose-1 more preferably, under the situation that 6-diphosphatase or its homologue activity are enhanced, preferably, give fine chemicals and increase about 20% or more.

In one embodiment, be enhanced in e. coli k12 protein b0464 or its homologue (shown in Table II the 5th or 7 row, the 334th row) activity, for example possible transcription repression albumen mtrr superfamily protein active is enhanced, preferably participate in transcribing control and/or DNA bonded protein active is enhanced, more preferably under the situation that the transcription repression albumen of multiple medicines efflux pump (TetR/AcrR family) or its homologue activity are enhanced, preferably, give each fine chemicals be increased in about 35% and 366% or higher between.

In one embodiment, be enhanced in e. coli k12 protein b1343 or its homologue (shown in Table II the 5th or 7 row, the 335th row) activity, the protein active that for example participates in rRNA processing and/or translation is enhanced, preferably under the situation that the ATP dependenc RNA helicase of 23S rRNA stimulation or its homologue activity are enhanced, preferably, give each fine chemicals be increased in about 38% and 51% or higher between.

In one embodiment, (be listed as at e. coli k12 protein b2414 or its homologue as Table II the 5th or 7, shown in the 336th row) activity is enhanced, for example the protein active of threonine dehydra(ta)se superfamily is enhanced, it is synthetic preferably to participate in amino acid bio, the biosynthesizing of halfcystine aromatic series, the degraded of halfcystine die aromatischen Aminosaeuren, the nitrogen and the sulphur of aspartate family utilize biosynthesizing, aspartic acid group amino acid degradation, sulfuric acid and the biosynthesizing of L-cysteine derivative, biosynthesizing from elementary amino acid whose secondary species, from glycine, the biosynthesizing of the secondary species of L-Serine and L-L-Ala, pyridoxal phosphate bonded protein active is enhanced, preferably under the situation that PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue activity are enhanced, preferably, give each fine chemicals be increased in about 37% and 75% or higher between.

In one embodiment, (be listed as at e. coli k12 protein b2762 or its homologue as Table II the 5th or 7, shown in the 337th row) activity is enhanced, for example 3 '-adenosine phosphate 5 '-phosphinylidyne sulfate reduction enzyme superfamily protein active is enhanced, preferably participate in C-compound and carbohydrate metabolism, C-compound and carbohydrate utilization, energy, glycolysis-and gluconeogenesis, plastid and/or photosynthetic protein active are enhanced, fructose-1 more preferably, under the situation that 6-diphosphatase or its homologue activity are enhanced, preferably, give fine chemicals and increase about 20% or more.

[0046.0.0.0] in one embodiment, under the situation that yeast saccharomyces cerevisiae protein YLR375W or its homologue activity are enhanced, preferably, giving fine chemicals and shikimic acid increases.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YBL015w or its homologue such as Ach1p is enhanced, preferably, giving fine chemicals and another kind of amino acid such as L-Ala increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173w or its homologue, for example participate in dna damage and reduction division pachytene stage check position activatory check position protein; Rad17p-Mec3p-Ddc1p is loaded into subunit or Rad24p or its homologue of the clip loading bin of DNA, under the situation as people or schizosaccharomyces pombe Rad17 raising, preferably, giving fine chemicals and another kind of amino acid such as leucine increases.

In one embodiment, under the situation that peroxysome matrix lipase or its homologue activity of yeast saccharomyces cerevisiae protein YOR084w or supposition is enhanced, preferably, giving fine chemicals or β-Gu Zaichun increases.

In one embodiment, be enhanced at e. coli k12 protein b1829 or its homologue activity, for example protease activity is enhanced, preferably the heat shock protein activity is enhanced, more preferably under the situation that htpX protein or its homologue activity are enhanced, preferably, giving fine chemicals and another amino acid such as phenylalanine increases.

In one embodiment, be enhanced at e. coli k12 protein b0464 or its homologue activity, for example under the situation that the transcription repression albumen of multiple medicines efflux pump (TetR/AcrR family) or its homologue activity are enhanced, preferably, give fine chemicals and other amino acid increase.

In one embodiment, be enhanced at e. coli k12 protein b1343 or its homologue activity, for example under the situation that the ATP dependenc RNA helicase of 23S rRNA stimulation or its homologue activity are enhanced, preferably, give fine chemicals and other amino acid increase.

In one embodiment, be enhanced at e. coli k12 protein b2414 or its homologue activity, for example under the situation that PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit activity are enhanced, preferably, give fine chemicals and other amino acid increase.

In one embodiment, activity at e. coli k12 protein b2762 or its homologue is enhanced, for example under the situation that 3 '-adenosine phosphate 5 '-phosphinylidyne sulfuric acid (PAPS) reductase enzyme or its homologue activity are enhanced, preferably, give fine chemicals and other amino acid increase.

In one embodiment, be enhanced, for example under the situation that fructose-1 or its homologue activity are enhanced, preferably, give fine chemicals and other amino acid increase at e. coli k12 protein b4232 or its homologue activity.

[0047.0.0.0] is in context, cell (preferred tissue, more preferably biological, as plant or microorganism or its part) in the quantity of each fine chemicals increase by 3% or higher, preferred especially 10% or higher, very particularly preferably more than 30% and most preferably 70% or higher, as 100%, 300% or 500%.

[0048.0.0.0] each fine chemicals can free form and/or is contained in the biology with protein or polypeptide or its mixture bonded form.Therefore, in one embodiment, the quantity of free form increases by 3% or higher in the cell (preferred tissue, more preferably biological as plant or microorganism or its part), preferred especially 10% or higher, very particularly preferably be higher than 30% and most preferably 70% or higher, as 100%, 300% or 500%.Therefore, in another embodiment, the quantity of each fine chemicals of bonded increases by 3% or higher in the cell (preferred tissue, more preferably biological as plant or microorganism or its part), preferred especially 10% or higher, very particularly preferably be higher than 30% and most preferably 70% or higher, as 100%, 300% or 500%.

[0049.0.0.0] has to give and improves each fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, the polypeptide of consensus sequence shown in the capable or 334-338 of 1-5 is capable, perhaps as Table II the 5th or 7 row, its function homologue described in polypeptide or the literary composition shown in the capable or 334-338 of 1-5 is capable, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the capable or 334-338 of 1-5 is capable) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.0.0] for the purposes of the present invention, term " L-methionine(Met) ", " methionine(Met) ", " homocysteine ", " S-adenosylmethionine " and " Threonine " also comprise corresponding salt, for example methionine hydrochloride or methionine sulfate.Preferably, term methionine(Met) or Threonine are intended to comprise term L-methionine(Met) or L-Threonine.

[0051.0.0.0] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example the fine chemicals composition.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce multiple the fine chemicals composition or the mixture of (as comprising other different aminoacids, lipid acid, VITAMIN, hormone, sugar, lipid or the like).

[0052.0.0.0] term " expression " is meant encoding gene fragment or gene transcription and/or translation.Usually, resulting product is mRNA or protein.Yet expression product also can comprise functional r NA, as antisense strand nucleic acid, tRNA, snRNA, rRNA, RNAi, siRNA, ribozyme etc.Expression can be whole, partial or instantaneous, for example is confined to particular cell types, tissue, organ or time phase.

[0053.0.0.0] in one embodiment, method of the present invention comprises one or more following steps:

A) stabilizing protein, described protein give the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) express and increase, have the activity of the raising fine chemicals described in the literary composition;

B) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of the raising methionine(Met) described in the literary composition expresses and improves;

C) improve the protein specific activity, described protein give the raising methionine(Met) that has described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) express and improve, perhaps reduce polypeptide of the present invention or be used for the inhibition regulation and control of the polypeptide of the inventive method;

D) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give the raising methionine(Met) that has described in the literary composition active, by coded protein or the polypeptide of the present invention of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or be used for the inventive method polypeptide (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) express and improve;

E) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give the raising methionine(Met) that has described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) express and improve;

F) express the transgenosis of coded protein, described protein give the raising methionine(Met) that has described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) express and improve;

G) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have the raising methionine(Met) described in the literary composition active, by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or be used for the inventive method polypeptide (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide);

H) by add positive Expression element or remove negative Expression element improve code book invention polypeptide or be used for the inventive method polypeptide (for example have as Table II the 3rd row, 1-5 is capable or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

I) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) (heat shock protein(HSP) for example of the present invention) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

J) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.0.0] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improve coded protein or have as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in) give methionine(Met) after active expression of polypeptides or the activity and increase.

[0055.0.0.0] is common, and the quantity of mRNA or polypeptide is relevant with coded proteinic quantity in biomass cells or the compartment, and therefore relevant with the overall activity of coded protein in the described volume.Described association is not always linear, and the activity in the volume depends on the stability of molecule or activates or suppress the existence of cofactor.In addition, product or educt are well-known to the inhibition of enzyme, and describe to some extent in textbook, as Stryer " Biochemistry ".

[0056.0.0.0] is common, and the quantity of mRNA, polynucleotide or nucleic acid molecule is relevant with coded proteinic quantity in biomass cells or the compartment, and therefore relevant with the overall activity of coded protein in the described volume.Described association is not always linear, and the activity in the volume depends on the stability of molecule, the degraded of molecule or the existence of activation or inhibition cofactor.In addition, product or educt are well-known to the inhibition of enzyme, for example Zinser etc. " Enzyminhibitoren/Enzymeinhibitors ".

[0057.0.0.0] can improve the protein of above being mentioned of nucleic acid molecule encoding of the present invention and/or the activity of polypeptide by number of ways.For example, by improving the activity that gene product quantity is increased in biology or its part (in cell), for example improve translation rate, and/or improve the stability of gene product and reduce protein degradation by introducing the stability that strong promoter improves expression rate or improve the mRNA that expresses.In addition, can influence the active or conversion of enzyme in the mode that reduces or increase speed of reaction or change (reduce or improve) substrate avidity.Polypeptide of the present invention or the sudden change of catalytic center that is used for the polypeptide (as enzyme) of the inventive method can be adjusted the turnover ratio of enzyme, for example indispensable amino acid knocks out the reduction that can cause enzymic activity or loses fully, or the disappearance of regulon binding site or sudden change can reduce negative the adjusting, as feedback inhibition (perhaps substrate suppresses, if the substrate level also improves).The specific activity that can improve enzyme of the present invention is to improve the combination of turnover ratio or promotion cofactor.Improving coding mRNA or proteinic stability also can increase the activity of gene product.Active stimulation also belongs to the scope of term " activity of raising ".

[0058.0.0.0] in addition, thereby the adjusting that can modify the aforementioned nucleic acid sequence improves genetic expression.This can regulate sequence or advantageously realize by modifying (for example sudden change) existing natural adjusting sequence by allos.These advantageous method also can make up mutually.

[0059.0.0.0] is common, the activity of gene product can improve by the quantity that improves coding mRNA specific in described biology or its part or respective egg white matter in biology or its part, especially vegetable cell, plant or plant tissue or its part or the microorganism." quantity of protein or mRNA " is interpreted as the molecule number of polypeptide in biology, tissue, cell or the cellular compartment or mRNA molecule." increase " of protein quantity is meant with wild-type, contrast or with reference to comparing, described protein molecular amounts in biology, tissue, cell or cellular compartment or its part quantitatively increases by for example one of following method.

The increase of [0060.0.0.0] molecular amounts preferably at least 1%, preferably more than 10%, more preferably 30% or more, preferred especially 50%, 70% or more, preferred especially especially 100%, most preferably 500% or more.Yet, from the beginning express and also be considered as theme of the present invention.

[0061.0.0.0] can cause modification (promptly improve or reduce) by endogenous or extrinsic factor.For example, active raising can cause by add gene product or precursor or activator or agonist in substratum or nutrition in biological or its part, or by instantaneous to biology or stably introduce described research object and cause.

[0062.0.0.0] realizes the raising or the reduction of plant or its part (as cell, tissue, organ, organoid etc.) fine chemicals quantity in one embodiment by the endogenous levels that improves polypeptide of the present invention or be used for the polypeptide of the inventive method.Therefore, transcribe and translate the endogenous level that adjusting can increase described polypeptide of the present invention or be used for the polypeptide of the inventive method by modified polypeptide.

[0063.0.0.0] in one embodiment can be by the orientation of native gene of the present invention or the fine chemicals quantity in random mutagenesis raising biology or its part.For example can use homologous recombination in promotor, to introduce positive regulatory element, or remove straining element from regulatory region as plant 35S enhanser.In addition, can use (Plant Physiol.2003 May as Kochevenko and Willmitzer; 132 (1): 174-84) and wherein the gene conversion of the described method of quoted passage is destroyed straining element or is strengthened the activity of positive regulatory element.

In addition, can positive element be introduced at random (plant) genome, and the screening positive element is incorporated near the gene of the present invention and therefore make genetic expression enhanced strain of the present invention be by T-DNA or transposon mutagenesis.Hayashi etc. 1992 (Science 258:1350-1353) or Weigel etc., 2000 (Plant Physiol.122,1003-1013) and other quoted passage of wherein being quoted random integration activated plant gene by enhancer element has been described.The reverse genetics strategy of identifying near the insertion (finally being loaded with active element) of goal gene has been described in a plurality of cases, Krysan etc. for example, 1999 (Plant Cell 1999,11,2283-2290); Sessions etc., 2002 (Plant Cell2002,14,2985-2994); Young etc., 2001, (Plant Physiol.2001,125,513-518); Koprek etc., 2000 (Plant J.2000,24,253-263); Jeon etc., 2000 (Plant J.2000,22,561-570); Tissier etc., 1999 (Plant Cell 1999,11,1841-1852); Speulmann etc. 1999 (Plant Cell 1999,11,1853-1866).Briefly, collection material and prepare genomic dna from floral all plants of big T-DNA or transposon mutagenesis.Then according to as Krysan etc., 1999 (Plant Cell 1999,11,2283-2290) described in special process merge genomic dna.Then by of the combination of specific multi-PRC reaction screening-gene group dna library with detection insertional mutagenesis agent (for example T-DNA or transposon) and goal gene.Therefore, use the particular combinations of T-DNA or transposon border primer and gene specific primer in dna library, to carry out the PCR reaction.(PlantCell 1999,11,2283-2290) for the visible Krysan of rule of design of primers etc. 1999.The lower level dna library screened again cause identifying wherein being inserted into property of goal gene mutagenic compound destructive plant individual.

The enhancing of positive regulatory element or the destruction of negative regulatory factor or weaken also can be by common induced-mutation technique realization: the generation of chemical mutation and radiomutation population is routine techniques and is understood by the technician.The method that is used for plant is described at " Methods in Molecular Biology " the 82nd volume by Koorneef etc. 1982 and quoted passage wherein and Lightner and Caspar.These technology are induced the point mutation that can use in arbitrary known of identifying as the method for tilling (Colbert etc. 2001) usually.

Therefore, if modify the native gene (gene that especially contains nucleic acid molecule of the present invention) that coding is given the polypeptide of expression of polypeptides raising of the present invention by homologous recombination, tilling method or gene conversion, then expression level can be enhanced.

[0064.0.0.0] regulates sequence can effectively be connected and control its stability of transcribing and translate or control coding mRNA or marking protein or degraded with the coding region of endogenous protein.In order to modify and control expression, can change, add or revise promotor, UTR, splice site, processing signal, polyadenylation site, terminator, enhanser, inhibition, transcribe or the posttranslational modification site.For example, Hayashi etc., 1992 (Science 258:1350-1353) or Weigel etc., 2000 (PlantPhysiol.122,1003-1013) and other document descriptions of wherein quoting the random integration activated plant gene by enhancer element.For example, can be by replacing endogenesis promoter with stronger transgenosis promotor or providing 3 ' UTR replacement endogenous, the 3 ' UTR of high stability more to regulate and control the expression level of endogenous protein with not changing the coding region.In addition, can come regulatory transcription to regulate by introducing manual transcription factor as be shown in the examples.Alternative promotor, terminator and UTR have hereinafter been described.

[0065.0.0.0] also can improve activation, the polypeptide of the present invention with above-mentioned active endogenous polypeptide or the activation that is used for the polypeptide of the inventive method by introducing synthetic transcription factor (for example improve express or activity after give each fine chemicals and increase), described synthetic transcription factor and endogenous polypeptide of the present invention or be used for the inventive method or the coding region of the polypeptide of process of the present invention or its endogenous homologue encoding gene is combined closely and synthesizing property transcription factor activates it and transcribes.Can resolve the chimeric zinc finger protein that contains specific DNA binding domains and activation structure territory (for example VP16 structural domain of hsv).Specific binding domains can be in conjunction with the regulatory region of endogenous protein coding region.The expression of chimeric transcription factor in biological (particularly plant) causes the present invention or is used for the specifically expressing of the endogenous polypeptide (particularly its plant homologue) of the inventive method, see for example WO01/52620, Oriz, Proc.Natl.Acad.Sci.USA, 2002, the 99 volumes, 13290 or Guan, Proc.Natl.Acad.Sci.USA, 2002, the 99 volumes, 13296.

[0066.0.0.0] is in another embodiment of the inventive method, use wherein a kind of said gene or a kind of above-mentioned nucleic acid plant through following sudden change, i.e. sudden change makes the activity of coded gene product compare the influence that less is subjected to cytokine with mutein not, or unaffected fully.For example, well-known enzymic activity regulation mechanism is that substrate suppresses or feedback regulation mechanism.Introduce to replace, disappearance and add one or more bases, Nucleotide or amino acid whose method in the corresponding sequence and technical description in hereinafter listed corresponding paragraph and reference, Sambrook etc. for example, " Molecular Cloning ", Cold Spring Habour, NY, 1989.The computer software means that those skilled in the art can comprise the algorithm of identifying binding site and adjustment structure territory by use with the situation of sequence of nucleic acid molecules of the present invention or its expression product and this area relatively, by to nucleic acid molecule or the sudden change of protein drawing-in system and measure can cause that specific activity improves or unit volume (especially individual cells) in those adjustment structure territory and binding sites that suddenly change and identify regulon of activity raising.

[0067.0.0.0] therefore, it is favourable expressing the polypeptide (as using prokaryotic gene at eucaryon host) come from nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or the polypeptide of the present invention of evolution co-relation biology far away or to be used for the inventive method in biology, because under these circumstances, the regulation mechanism of host cell can reducer or the activity (cytoactive or specific activity) of its expression product.

[0068.0.0.0] be not can cause the mode of disadvantageous effect to introduce sudden change to amino acid whose generation.

The adjusting of [0069.0.0.0] gene or its gene product is subjected to less influence and is interpreted as enzymic activity is regulated decline, causes the specific activity of gene or its product or the raising of cytoactive.The activity that the raising of enzymic activity can be regarded as enzyme improves at least 10% with initial biophase ratio, advantageously is at least 20%, 30% or 40%, particularly advantageously is at least 50%, 60% or 70%.This causes that required each fine chemicals productivity improves.

[0070.0.0.0] since as described below will give nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention be used for the gene of expression of polypeptides of the inventive method or a plurality of gene (nucleic acid construct of mentioning for example) separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, the favorable amino acid composition that for example contains each fine chemicals of high level (from the physiology of nutrition angle) particularly is that amino acid equally also is fine chemicals.

[0071.0.0.0] preferably, with composition of the present invention or composition biological or that its part provides also contain higher amount to the nutrition of animal and human's class or healthy have the metabolite that just influencing or low quantity to nutrition of animal and human's class or healthy metabolite with negatively influencing.Similarly, can improve the number or the activity that input or output other required genes of nutrition or metabolite (comprising amino acid and precursor thereof that the cell biological synthesizing amino acid is required), to improve the concentration of necessity in cell or the corresponding storage compartment or associated precursors, cofactor or intermediate product.Because polypeptide of the present invention or be used for the raising or the active new generation of the polypeptide active of the inventive method, perhaps, can in host living beings such as plant or microorganism, improve amino acid whose output, production and/or generation efficient owing to nucleic acid sequence of the present invention and/or to participating in the raising of other generegulation of amino acid bio synthetic (activity of for example passing through to improve the activity of the enzyme that synthesizes precursor or participating in one or more genes of amino acid decomposition by destruction).

[0072.0.0.0] therefore might produce the more favourable sulfocompound that contains at least one covalent attachment sulphur atom in the methods of the invention by influencing metabolism.The example of this compounds also has homocysteine, S-adenosylmethionine, halfcystine except methionine(Met), be preferably methionine(Met) and S adenosylmethionine.

[0073.0.0.0] therefore, in one embodiment, method of the present invention relates to and comprises following method:

A) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

B) improve polypeptide of the present invention or be used for the polypeptide of the inventive method or its homologue (for example Table II the 5th or 7 row, 1-5 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of promptly giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

C) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

D) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded, and optional other dissociates and/or in conjunction with amino acid by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis.

[0074.0.0.0] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded or free and bonded fine chemicals, if and dissociate or/and in conjunction with amino acid Methionin particularly as selecting to produce, reclaim and expect to separate other.People such as Galili, Transgenic Res., 200,9,2,137-144 has described, and heterogenous expression is used for amino acid bio synthetic bacterial gene can make free and the increase of protein bound amino acid.

[0075.0.0.0] (also is included in generation activity in the biology as the front definition through aforesaid increase, promptly from the beginning active) afterwards, for example after nucleic acid molecule described in introducing and expression nucleic acid molecule of the present invention or the inventive method or the process, cultivating also and gather in the crops biology of the present invention subsequently, advantageously is microorganism, non-human animal, plant, plant or animal tissues or plant or zooblast.

[0076.0.0.0] is fit to according to the present invention and the biology or the host living beings (genetically modified organism) of nucleic acid molecule, nucleic acid construct or the carrier (all seeing below) that use for the inventive method are all biologies that can synthesize each fine chemicals and suitable activation, introducing or stimulated gene in principle.The example that can mention is plant, microorganism such as fungi, bacterium, yeast, algae or a diatom genetically modified or that obtain by directed mutagenesis or random mutagenesis and binding specificity chosen process.Preferred biology is naturally to synthesize the biology of each fine chemicals in a large number, as fungi, yeast or plant.Say that in principle transgenic animal such as Caenorhabditis elegans (Caenorhabditis elegans) also are suitable for as host living beings.

[0077.0.0.0] when genetically modified organism be microorganism, for example eukaryote such as fungi, algae, when diatom or yeast, particularly be selected from chaetomium section (Chaetomiaceae), the mould section of hairpin (Choanephoraceae), Cryptococcaceae (Cryptococcaceae), Cunninghamellaceae (Cunninghamellaceae), the dark born of the same parents sections (Demetiaceae) that obstruct, Moniliaceae (Moniliaceae), Mortierellaceae (Mortierellaceae), Mucoraceae (Mucoraceae), pythiaceae (Pythiaceae), Saccharomycetaceae, Saprolegniaceae (Saprolegniaceae), fission yeast section (Schizosaccharomycetaceae), Sodariaceae section, Sporobolomycetaceae (Sporobolomycetaceae), Tuberculariaceae (Tuberculariaceae), Adelotheciaceae section, Dinophyceae (Dinophyceae), the fungi of ox hair moss section (Ditrichaceae) or green branch algae guiding principle (Prasinophyceae), algae, diatom or yeast, or prokaryotic organism, as bacterium or blue-green algae, Actinomy cetaceae (Actinomycetaceae) particularly, Bacillaceae (Bacillaceae), Brevibacteriaceae (Brevibacteriaceae), rod Bacteriaceae (Corynebacteriaceae), enterobacteriaceae, Gordon Salmonella section (Gordoniaceae), Nocardiaceae (Nocardiaceae), micrococcaceae (Micrococcaceae), mycobacteriaceae (Mycobacteriaceae), pseudomonadaceae (Pseudomonaceae), when Rhizobiaceae (Rhizobiaceae) or Streptomycetaceae (Streptomycetaceae), cultivate this biology on solid or liquid nutrient medium, described substratum is known and be suitable for this biology for the technician.Later can gather in the crops biology in vegetative period.

[0078.0.0.0] can directly be processed into (isolating in case of necessity) each fine chemicals of microorganism or recovery food or animal-feed or be used for other application subsequently, for example carries out according to EP-B-0533039 or the disclosed content of EP-A-0615693 (this paper is incorporated herein by reference especially).Can be by extracting and precipitation or by ion-exchanger with those skilled in the art are known and hereinafter described additive method, purified fermentation broth or tunning in a usual manner.These different products of steps of checking are amino acid or still contain fermented liquid and the amino-acids composition of different quantities cellular component, and the favourable fermented liquid and the scope of cellular component are 0 to 99% by weight, preferably are lower than 80%, especially preferably are lower than 50%.

[0080.0.0.0] particularly preferred bacterial strain is for being selected from Bacillaceae, Brevibacteriaceae, the rod Bacteriaceae, Nocardiaceae, mycobacteriaceae, Streptomycetaceae, the enterobacteriaceae bacterial strain, Bacillus circulans for example, subtilis (Bacillus subtilis), bacillus certain (Bacillus sp.), Brevibacterium albidum, Brevibacterium album, Brevibacteriumcerinum, brevibacterium flavum (Brevibacterium flavum), the Brevibacteriumglutamigenes brevibacterium iodinum, Brevibacterium ketoglutamicum, brevibacterium lactofermentum (Brevibacterium lactofermentum), extension brevibacterium, Brevibacteriumroseum, Brevibacterium saccharolyticum (Brevibacterium saccharolyticum), brevibacterium sp certain (Brevibacterium sp.), Corynebacterium acctoacidophlum (Corynebacteriumacetoacidophilum), Corynebacterium acetoglutamicum, produce ammonia rod bacillus, Corynebacterium glutamicum (=micrococcus glutamicus (Micrococcus glutamicum)), corynebacterium melassecola (Corynebacterium melassecola), Corynebacterium certain (Corynebacterium sp.), purplish red Nocardia bacteria (Nocardia rhodochrous) (prunosus red coccus (Rhodococcusrhodochrous)), red thick plywood bacillus (Mycobacterium rhodochrous), muta lead mycillin (Streptomyces lividans) and intestinal bacteria (Escherichia coli), especially e. coli k12.

[0081.0.0.0] in addition, particularly preferred bacterial strain is for being selected from Cryptococcaceae, Saccharomycetaceae, fission yeast section, mycocandida for example, Chinese Sen Shi yeast belong, Pichia, the bacterial strain of yeast belong and Schizosaccharomyces, preferred bacterial strain is selected from Rhodotorula rubra HMC, rhodotorula glutinis, herbage rhodotorula (Rhodotorula graminis), separate the inferior sieve yeast of fat, reddish brown shadow yeast, Sporobolomycesshibatanus, yeast saccharomyces cerevisiae, Candida boidinii, Candida bombicola, the column candiyeast, Candida parapsilosis, fold candida, candida tropicalis, pichia methanolica and pichia pastoris phaff.

All above-mentioned biologies also can be used as host living beings in principle and work.

[0083.0.0.0] particularly preferred plant is to be selected from following plant: composite family, for example Helianthus, Tagetes, for example Sunflower Receptacle (Helianthus annuus) [Sunflower Receptacle (sunflower)], spiceleaf Flower of Aztec Marigold (Tagetes lucida), Flower of Aztec Marigold (Tagetes erecta) or Tagetes signata (Tagetestenuifolia) [Flower of Aztec Marigold (Marigold)]; Brassicaceae, for example Btassica, Arabidopsis, for example colea (Brassica napus), overgrown with weeds blue or green (Brassica rapa ssp.) [rape (canola), rape (oilseed rape), turnip rape] or Arabidopis thaliana (Arabidopsis thaliana); Pulse family (Fabaceae), for example Glycine, for example soybean (Glycine max), Soja hispida or Sojamax [soybean (soybean)]; Flax family (Linaceae), for example linum, for example flax (Linumusitatissimum) [flax (flax), linseed oil (linseed)]; Gramineae, for example Hordeum, Secale, Avena, sorghum, Oryza, Zea, Triticum, for example barley (Hordeumvulgare) [barley (barley)]; Rye (Secale cereale) [rye (rye)], oat (Avenasativa), wild avena sativa (Avena fatua), than praising oat (Avena byzantina), the former mutation of wild avena sativa (Avena fatua var.Sativa), hybrid oat (Avena hybrida) [oat (oat)], dichromatism chinese sorghum (Sorgh um bicolor) [Chinese sorghum (Sorghum), millet (millet)], rice (Oryza sativa), broad-leaved wild-rice (Oryza latifolia) [rice (rice)], Zea mays (Zea mays) [corn (corn), corn (maize)], common wheat (Triticum aestivum), durum wheat (Triticum durum), cylinder wheat (Triticum turgidum), Triticum hybern um, agate card wheat (Triticummacha), common wheat (Triticum sativum) or common wheat (Triticum vulgare) [wheat (wheat), bread wheat (bread wheat), common wheat (common wheat)]; Solanaceae, for example Solanum, tomato belong to, for example potato (Solanum tuberosum) [potato (potato)], tomato (Lycopersicon esculentum), Lycopersicon lycopersicum, Lycopersiconpyriforme, red eggplant (Solanum integrifolium) or tomato (Solanum lycopersicum) [tomato (tomato)].

[0084.0.0.0] all above-mentioned biologies also can be used as host living beings in principle.

[0085.0.0.0] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps (c) (a) with (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.0] therefore, the purposes that nucleotide sequence of the present invention or nucleic acid construct of the present invention are used to produce transgenic plant also is a theme of the present invention.

[0087.0.0.0] can be separated in each fine chemicals of synthetic in biology of the present invention, particularly microorganism, cell, tissue or the plant in case of necessity.As hereinafter describing, depend on the purposes of each fine chemicals, can advantageously obtain different purity by purifying.

[0088.0.0.0] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified aminoacids content.Often be subject to minority indispensable amino acid (as Methionin, Threonine or methionine(Met)) owing to for example be used to raise the nutritive value of the plant of monogastric animal, so this is very important for the plant breeder.

[0088.1.0.0] in one embodiment, after improving or having produced polypeptide of the present invention or be used for the activity of polypeptide of the inventive method, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, the transgenic plant that produced can grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

The plant that [0089.0.0.0] is as described below or its part (as leaf, root, flower and/or stem) and/or other can gather in the crops material can be directly as food or animal-feed or further process.In addition, can pass through extraction and precipitation or, be further purified amino acid in a usual manner by ion-exchanger and other methods well known by persons skilled in the art and hereinafter described.Be suitable for multiple application and be the amino-acids composition of amino acid or the other plant component that contains different quantities by the product that these different processing methods obtain, described other components favourable for by weight from 0 to 99%, preferably be lower than 90%, especially preferably be lower than 80%.Plant can also advantageously not carry out other processing and directly uses, as feed or be used for extracting.

[0090.0.0.0] chemical pure each fine chemicals or the chemical pure composition that contains each fine chemicals can produce by preceding method.For this reason, from the substratum of biology of the present invention (as microorganism, non-human animal or plant) and/or cultivation biology, separate each fine chemicals or composition in known manner.These chemical pure each fine chemicals or described composition are favourable for the application in foodstuffs industry, cosmetic industry or the pharmaceutical industry.

[0091.0.0.0] therefore, the plant component foreign matter content is low as far as possible, and preferably other foreign matter contents are also low as far as possible, and aforementioned each fine chemicals that obtains is pure form as far as possible.In these are used, the content of plant component favourable for by weight less than 10%, preferred 1%, more preferably 0.1%, very particularly preferably 0.01% or lower.

[0092.0.0.0] therefore, each fine chemicals purity that the present invention produces by weight at least 0.1%, preferably is higher than 1%, more preferably 10% even more preferably be higher than 50%, 60%, 70% or 80% even preferably be higher than 90%, most preferably be 95%, 99% or higher.

[0093.0.0.0] in this article, compare with wild-type, contrast or reference, the quantity of each fine chemicals can improve at least 1.1 times according to the inventive method, preferably at least 1.5,2 or 5 times in the cell of the present invention, especially preferably at least 10 or 30 times, very particularly preferably at least 50 times.Preferably, described raising is present in the tissue, more preferably is present in biology or its part that can gather in the crops.

[0094.0.0.0] says in principle, can increase each fine chemicals that is produced in two ways by method of the present invention.Each fine chemicals of each fine chemicals content, particularly protein bound of each each fine chemicals of fine chemicals pond, particularly free of raising free that can be favourable and/or protein bound.

[0095.0.0.0] advantageously increases the total free aminoacids pond to separate a large amount of each pure fine chemicals by method of the present invention in genetically modified organism.

[0096.0.0.0] is in another embodiment preferred of the present invention, improving nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide (as amino acid needed pond as methionine(Met), Methionin or Threonine in the biology) combines and can be used for producing each fine chemicals and (consult US5,589,616, WO 96/38574, WO 97/07665, WO 97/28247, US 4,886,878, US 5,082,993 and US 5,670,635).Galili etc. (Transgenic Res.2000) prove that strengthening the synthetic free threonine that not only causes of Threonine by feedback non-sensitive type E.C. 2.7.2.4. increases, and causes that also the Threonine of protein bound increases.

Each fine chemicals content that [0097.0.0.0] improves protein bound also is favourable.

[0098.0.0.0] in preferred embodiments, each fine chemicals (methionine(Met)) and/or Threonine be produce according to the present invention and carry out isolating where necessary.It is favourable as Methionin and aminoacid mixture that the method according to this invention produces other amino acid.

For microbial fermentation, aforementioned amino acid can be accumulated in substratum and/or the cell [0099.0.0.0].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Can handle spissated fermented liquid by freeze-drying, spraying drying, spraying choosing grain or additive method subsequently.

[0100.0.0.0] is for purifying amino acid, can use suitable resin that the fermented liquid that contains product of having removed biomass is carried out chromatogram, described resin such as ion exchange resin, as negatively charged ion or Zeo-karb, hydrophobic resin or hydrophilic resin for example Resins, epoxy, polyurethane resin or polyacrylamide resin, or according to the isolating resin of the molecular weight of compound, as polyvinyl chloride homopolymer resin or form by for example vinylformic acid polymer, with the crosslinked resin of polyenoid ether or divinyl ethylene glycol as

,

With

Necessary, can use identical or other chromatography resin repeats these chromatographic steps.The technician is familiar with selection and their the most effective purposes of suitable chromatography resin.The product of purifying can and be stored under the temperature of guaranteeing the product maximum stable by filtration or ultrafiltration and concentration.

[0101.0.0.0] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) BioprocessEngineer.19:67-70. " Ulmann ' s Encyclopedia of Industrial Chemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology ", John Wiley andSons; Fallon, A. etc. (1987) " Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology ", the 17th volume.

[0102.0.0.0] amino acid can be for example as (Plant Cell﹠amp such as Geigenberger; Environ, 19,1996:43-55) describedly in alcohol extract, detect by HPLC convenient separation ground.Can use hot-water extraction amino acid.Filter the back and dilute extract with the water that contains 20mg/mL sodium acetate (sodium acide).Use anion-exchange column to separate and detect amino acid with electrochemical detector.Ins and outs can be taken from Y.Ding etc., 2002, Direct determination of free amino acids andsugars in green tea by anion-exchange chromatography with integratedpulsed amperometric detection, J Chromatogr A, (2002) 982; 237-244, or take from for example Karchi etc., 1993, Plant is J.3:721-727; Matthews MJ, 1997 (Lysine, threonine and methionine biosynthesis.BK Singh compiles, " PlantAmino Acids:Biochemistry and Biotechnology ", Dekker, New York, 205-225 page or leaf; H Hesse and R Hoefgen. (2003) Molecular aspects ofmethionine biosynthesis.TIPS 8 (259-262).

[0103.0.0.0] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

A) preferred nucleic acid molecule or its fragment of encoding mature form polypeptide at least, described polypeptide has as Table II the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable, and described nucleic acid molecule gives that each fine chemicals quantity increases in biology or its part;

B) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

G) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

H) nucleic acid molecule, its comprise by use have as Table III the 7th row, 1-5 is capable and/or 334-338 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

I) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

J) encoded polypeptides comprise have as Table IV the 7th row, 1-5 is capable and/or 334-338 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

K) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the structural domain of polypeptide; With

L) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0103.1.0.0] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II A the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide of sequence.

[0103.2.0.0] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule IIB the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide of sequence.

[0104.0.0.0] in one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention or the nucleic acid that is used for the inventive method not by Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide of sequence.

[0105.0.0.0] unless otherwise indicated, term " polynucleotide ", " nucleic acid " and " nucleic acid molecule " are used interchangeably in the context of the invention.Unless otherwise indicated, term " peptide ", " polypeptide " and " protein " are used interchangeably in the context of the invention.Term " sequence " can relate to polynucleotide, nucleic acid, nucleic acid molecule, peptide, polypeptide and protein, and this depends on the linguistic context of using term " sequence ".Term used herein " gene ", " polynucleotide ", " nucleotide sequence ", " nucleotide sequence " or " nucleic acid molecule " are meant the polymer form Nucleotide of random length, can be Yeast Nucleic Acid or thymus nucleic acid.Term only relates to the primary structure of molecule.

[0106.0.0.0] therefore, term used herein " gene ", " polynucleotide ", " nucleotide sequence ", " nucleotide sequence " or " nucleic acid molecule " comprise double-stranded and single stranded DNA and RNA.The modification that it also comprises known type, for example methylate, " adding cap ", substitute one or more naturally occurring Nucleotide with analogue.Preferably, DNA of the present invention or RNA sequence contain the encoding sequence of the polypeptide defined herein of encoding.

[0107.0.0.0] " encoding sequence " is meant when placing the suitable adjustable sequence to control the nucleotide sequence that following time is transcribed into mRNA and/or is translated as polypeptide.The border of encoding sequence is by 5 ' terminal translation initiation codon and 3 ' terminal translation stop codon decision.Encoding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequence or genomic dna, wherein also can have intron under some environment.

Advantageously improved in [0108.0.0.0] method of the present invention and had as Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in the capable and/or 334-338 of 1-5 is capable, from being listed as Table II the 5th or 7, aminoacid sequence shown in the capable and/or 334-338 of 1-5 is capable is derived or from containing just like Table IV the 7th row, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in the capable and/or 334-338 of 1-5 is capable, perhaps its coding has as Table II the 3rd, 5 or 7 row, active or the bioactive polypeptide of polypeptidase shown in the capable and/or 334-338 of 1-5 is capable or for example give derivative or the homologue that each fine chemicals increases behind its expression or active the increasing.

[0109.0.0.0] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make each fine chemicals that the inventive method produced be optimized synthetic.

[0110.0.0.0] help the inventive method and coding have polypeptide of the present invention be used for the polypeptide of the inventive method or be used for process of the present invention polypeptide (for example Table II the 5th row, 1-5 is capable and/or 334-338 capable shown in protein or by Table I the 5th row, 1-5 is capable and/or 334-338 capable shown in coded protein or its homologue of nucleic acid molecule, as Table II the 7th row, 1-5 is capable and/or 334-338 capable shown in) nucleic acid molecule of active polypeptide can determine from generally open database.

[0111.0.0.0] must should be mentioned that common gene database in this article especially, as EMBL database (Stoesser G. etc., Nucleic Acids Res 2001, the 29 volume, 17-21), GenBank database (Benson D.A. etc., Nucleic Acids Res 2000, the 28th volume, 15-18) or PIR database (Barker W.C. etc., Nucleic Acids Res.1999, the 27th volume, 39-43).Can also use the biologic specificity gene database to determine favourable sequence, advantageously use for example SGD database (Cherry J.M. etc. for yeast, Nucleic Acids Res.1998, the 26th volume, 73-80) or MIPS database (Mewes H.W. etc., Nucleic Acids Res.1999, the 27th volume, 44-48), be GenProtEC database (http://web.bham.ac.uk/bcm4ght6/res.html) for intestinal bacteria, be TAIR database (Huala for Arabidopis thaliana, E. etc., Nucleic Acids Res.2001, the 29th (1) volume, 102-5) or the MIPS database.

The nucleic acid molecule that uses in [0112.0.0.0] the inventive method is the isolated nucleic acid sequences form, its coding have as Table I the 3rd row, 1-5 is capable and/or 334-338 capable shown in polypeptide active polypeptide or have as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the polypeptide of peptide sequence, and give each fine chemicals and increase.

[0113.0.0.0] is used for advantageously deriving from eukaryote at the nucleotide sequence of each fine chemicals of genetically modified organism generation, but also can derive from prokaryotic organism or archeobacteria, so it can be from for example microorganism, animal or plant.

[0114.0.0.0] for the purposes of the present invention, usually plural number is intended to comprise odd number and vice versa.

[0115.0.0.0] is integrated into nucleotide sequence in nucleic acid construct and/or the carrier in order to improve the importing of nucleotide sequence and the expression of sequence in the used genetically modified organism in method.The sequence of in the inventive method as herein described, using, also can additionally there be other nucleotide sequences in nucleic acid construct or carrier and import together in the biology, and described nucleotide sequence advantageously is the nucleotide sequence of the biosynthesis gene of each fine chemicals that the inventive method produced.Yet these extra sequences also can import in the biology by other independent nucleic acid constructs or carrier.

[0116.0.0.0] uses cloning vector mentioned in this article and method for transformation, as publishing and be incorporated in " Plant Molecular Biology and Biotechnology " (CRC Press, Boca Raton, Florida), the 6/7th chapter, 71-119 page or leaf, (1993); F.F.White, " Vectors for GeneTransfer in Higher Plants " " Transgenic Plants ", the 1st volume, Engineeringand Utilization, Kung and R.Wu compile, Academic Press, 1993,15-38; B.Jenes etc., Techniques for Gene Transfer, " Transgenic Plants ", and the 1st volume, Engineering and Utilization, Kung and R.Wu compile, Academic Press (1993), 128-143; Potrykus, Annu.Rev.Plant.Physiol.Plant Molec.Biol.42 (1991), carrier 205-225)) and method for transformation and the additive method of hereinafter quoting, nucleic acid can be widely used in the recombinant modified of biology (particularly prokaryotic organism or eukaryotic microorganisms or plant), so they become the better and more effective producer of each fine chemicals that the inventive method produces.Can cause each fine chemicals or from their product (as the protein of modifying) improved production or production efficiency by the direct effect of operation or the indirect effect by this operation.

[0117.0.0.0] in one embodiment, nucleic acid molecule of the present invention derives from plant, as be selected from Aceraceae (Aceraceae), Anacardiaceae, umbelliferae, composite family, brassicaceae, Cactaceae (Cactaceae), Curcurbitaceae, Euphorbiaceae, pulse family (Fabaceae), Malvaceae, Nymphaeceae (Nymphaeaceae), papaveracease, the Rosaceae (Rosaceae), Salicaceae (Salicaceae), Solanaceae, Palmae (Arecaceae), Bromelia family, Cyperaceae (Cyperaceae), Iridaceae (Iridaceae), Liliaceae (Liliaceae), the orchid family (Orchidaceae), Gentianaceae (Gentianaceae), Labiatae (Labiaceae), Magnoliaceae (Magnoliaceae), Ranunculaceae (Ranunculaceae), Carifolaceae, Rubiaceae, scrophulariaceae, Caryophyllaceae (Caryophyllaceae), Ericaceae, polygonaceae (Polygonaceae), Violaceae (Violaceae), the plant of rush family (Juncaceae) or Gramineae (Poaceae), and preferably derive from and be selected from umbelliferae, composite family, brassicaceae, Curcurbitaceae, pulse family (Fabaceae), papaveracease, the Rosaceae, Solanaceae, the plant of Liliaceae or Gramineae (Poaceae).Preferably crop plants and the plant above mentioned as host plant, section As mentioned above and genus, for example preferred species cashew nut, Potmarigold Calendula, safflower, witloof, arithoke, Sunflower Receptacle, spiceleaf Flower of Aztec Marigold, Flower of Aztec Marigold, Tagetes signata; Radix Dauci Sativae; Wood-nut, Turkey hazel, Borrago officinalis; Colea; overgrown with weeds blue or green; wild Europe sinapsis alba; leaf mustard; the former mutation of leaf mustard; the wrinkle leaf mustard; leafy mustard; the black mustard dish; Brassica sinapioides; Melanosinapis communis; wild cabbage; Arabidopis thaliana; pineapple; Ananas ananas; Bromelia comosa; papaya; hemp; sweet potato; violin leaf morning glory; Convolvulusbatatas; Convolvulus tiliaceus; Ipomoea fastigiata; Ipomoea tiliacea; the trilobated leaf potato; Convolvulus panduratus; beet; beta vulgaris; the former mutation of beet; coastal beet; Beta vulgaris var.perennis; Beta vulgaris var.conditiva; Beta vulgarisvar.esculenta; winter squash; the opium pumpkin; summer squash; pumpkin; Fructus oleae europaeae; cassava; Janipha manihot; Jatropha manihot.; Manihot aipil; Manihot dulcis; Manihot manihot; Manihot melanobasis; Manihot esculenta; castor-oil plant; pea; feeding pea; Pisum humile; alfalfa; Yellow Sickle Medick; the hybridization clover; soybean; Dolichos soja; the climing beans of wide leaf; Glycine hispida; Phaseolus max; Sojahispida; Soja max; coconut; the currant Flos Pelargonii; Oleum cocoas; bay; avocado; Semen arachidis hypogaeae; flax; Linum humile; Austria flax; Linum bienne; narrowleaf flax; purging flaw; golden yellow flax; Da Hua flax; Adenolinum grandiflorum; Lewis flax; that other flax; Iinum peerenne L.; Linum perenne var.lewisii; Linum pratense; Linum trigynum; pomegranate; upland cotton; tree cotton; sea island cotton; cotton; plucked instrument Bai Shi cotton; banana; the wild any of several broadleaf plants of fruitlet; the powder bajiao banana; some kind of Musa; oil palm; the ghost opium poppy; the Flos Papaveris rhoeadis; Papaver dubium; flax; Piper aduncum; Piper amalago; matico; Ao Letu sets pepper; betel; Mountain Spicy Tree Fruit; long pepper; pepper; false Piper longum; Artanthe adunca; Artanthe elongata; Peperomia elongata; Piper elongatum; Steffensiaelongata; barley (Hordeum vulgare); the awns Hordeum jubatum; the beach barley; wild covered barley grass; the cultivation two rowed barley; Hordeum aegiceras; six-rowed barley; Hordeumhexastichum; Hordeum irregulare; barley (Hordeum sativum); wild covered barley grass; oat; wild avena sativa; than praising oat; the former mutation of wild avena sativa; the hybrid oat; dichromatism chinese sorghum; Johnson grass (Sorghum halepense); sweet sorghum; chinese sorghum; Andropogondrummondii; Holcus bicolor; Holcus sorghum; Sorghum aethiopicum; Sorghum arundinaceum; the Ka Foer jowar; fringe jowar grass hangs down; Sorghum dochna; Sorghum drummondii; hard jowar grass; Sorghum guineense; Sorghumlanceolatum; many arteries and veins jowar grass; sweet sorghum; Sorghum subglabrescens; Sorghumverticilliflorum; chinese sorghum; Johnson grass (Holcus halepensis); Sorghum miliaceummillet; Panicum militaceum; Zea mays; common wheat (Triticum aestivum); durum wheat; the cylinder wheat; Triticum hybernum; agate card wheat; common wheat (Triticumsativum) or common wheat (Triticum vulgare); some kind of Coffea; fruitlet coffee; middle fruit coffee; big fruit coffee; capsicum; capsicum glabriusculum mutation; hot millet; capsicum; tobacco; potato; eggplant; tomato; Lycopersicon lycopersicum; Lycopersiconpyriforme; red eggplant; tomato; cocoa tree or tea.

[0118.0.0.0] in one embodiment, be used for the microorganism in the sequence of nucleic acid molecules that microorganism produces each fine chemicals advantageously derives from above host living beings, mentioned, as fungi, as Aspergillus, Penicillium or Claviceps, or derive from yeast, as Pichia, torulopsis, Hansenula, Schizosaccharomyces, mycocandida, Rhodotorula or yeast belong, the special yeast that preferably advantageously derives from Saccharomycetaceae is as favourable yeast belong and particularly advantageous yeast saccharomyces cerevisiae.

[0119.0.0.0] technician understands other suitable sources that produce each fine chemicals, and described suitable source also has the nucleic acid molecule source of usefulness.They generally comprise all prokaryotic organism or eukaryotic cells, preferred unicellular microorganism, as fungi, as Claviceps or Aspergillus, perhaps gram-positive microorganism, belong to (Caseobacter) or genus arthrobacter as bacillus, Corynebacterium, micrococcus sp, brevibacterium sp, Rhod, Nocardia, butter bacillus, perhaps Gram-negative bacteria, for example Escherichia, Flavobacterium or salmonella, perhaps yeast is as Rhodotorula, Chinese Sen Shi yeast belong or mycocandida.

[0120.0.0.0] particularly advantageously, the production bacterial strain of selecting in the inventive method is to be selected from following microorganism: Actinomy cetaceae, Bacillaceae, Brevibacteriaceae, the rod Bacteriaceae, enterobacteriaceae, Gordon Salmonella section, micrococcaceae, mycobacteriaceae, Nocardiaceae, pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, chaetomium section, the mould section of hairpin, Cryptococcaceae, Cunninghamellaceae, the dark born of the same parents section that obstructs, Moniliaceae, Mortierellaceae, Mucoraceae, pythiaceae, Saccharomycetaceae, Saprolegniaceae, fission yeast section, Sodariaceae section, Sporobolomycetaceae, Tuberculariaceae, Adelotheciaceae section, Dinophyceae, ox hair moss section and green branch algae guiding principle or Hansenula anomala, Candida utilis, ergot (Claviceps purpurea), Bacillus circulans, Bacillus subtilus, bacillus certain (Bacillus sp.), Brevibacterium albidum, Brevibacterium album, Brevibacterium cerinum, brevibacterium flavum, Brevibacterium glutamigenes, brevibacterium iodinum, Brevibacterium ketoglutamicum, brevibacterium lactofermentum, extension brevibacterium, Brevibacterium roseum, Brevibacterium saccharolyticum, brevibacterium sp certain (Brevibacteriumsp.), Corynebacterium acctoacidophlum, Corynebacterium acetoglutamicum, produce ammonia rod bacillus, Corynebacterium glutamicum (=micrococcus glutamicus), corynebacterium melassecola, Corynebacterium certain (Corynebacterium sp.) or intestinal bacteria, particularly e. coli k12 and described bacterial strain thereof.

[0121.0.0.0] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in peptide sequence or its functional homologue as herein described the difference of one or more amino acid moleculars is arranged, described artificial sequence is preferably given aforementioned activity, and promptly giving each fine chemicals after improving its activity increases.

[0122.0.0.0] can use the nucleotide sequence that contains synthetic that (in due course) can be integrated into DNA or RNA, the non-natural or nucleotide base modified in the methods of the invention.Described synthetic base non-natural or that modify can for example improve nucleic acid molecule in the extracellular or intracellular stability.Nucleic acid molecule of the present invention can contain modification as hereinbefore.

The term " nucleic acid molecule " that uses in [0123.0.0.0] this paper context also can comprise the non-translated sequence that is positioned at encoding gene district 3 ' and 5 ' end, at least 100, preferred 50, preferred especially 20 Nucleotide in for example at least 500, preferred 200 of coding region 5 ' end upstream, preferred especially 100 Nucleotide and 3 ' end downstream, encoding gene district.Usually advantageously only select the coding region to be used to the purpose of cloning and expressing.

[0124.0.0.0] preferably, nucleic acid molecule that uses in the inventive method or nucleic acid molecule of the present invention are isolated nucleic acid molecule.

Other polynucleotide or the nucleic acid molecule that exist in [0125.0.0.0] " isolating " polynucleotide or nucleic acid molecule and the nucleic acid molecule natural origin are separated.Isolated nucleic acid molecule can be the chromosome segment of several kb, or is preferably the molecule that only contains gene coding region.Therefore, isolated nucleic acid molecule of the present invention can contain 5 ' and 3 ' adjacent chromosomal region or other adjacent chromosomal region, but does not preferably contain naturally occurring those sequences of biological gene group or karyomit(e) inclusion amplifying nucleic acid molecular sequences both sides that nucleic acid molecule originates (for example with coding nucleic acid molecule 5 ' and 3 ' UTR regional adjacent sequence).In multiple embodiments, the isolated nucleic acid molecule of using in the inventive method can contain the naturally occurring nucleotide sequence less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb in genomic dna amplifying nucleic acid molecule both sides of nucleic acid institute derived cell.

[0126.0.0.0] can use standard molecular biological technique and sequence information provided herein to be separated in the nucleic acid molecule that uses in the method, polynucleotide for example of the present invention or its part.For example, can also be by the homologous sequence or the homology conserved sequence district of comparison algorithm identification of dna or amino acid levels.The former can be at standard hybridization technique (Sambrook etc. for example, " Molecular Cloning:ALaboratory Manual " the 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, those described in 1989) be used for separating other useful nucleotide sequences of present method as probe in.

[0127.0.0.0] in addition, can separate the nucleic acid molecule of the complete sequence that comprises nucleic acid molecule that present method is used by the polymerase chain reaction, polynucleotide for example of the present invention or its part are wherein used the Oligonucleolide primers based on described sequence or its part.The nucleic acid molecule that for example contains complete sequence or its part can separate by the polymerase chain reaction of using the Oligonucleolide primers that produces based on this sequence.For example, can be from cell separating mRNA (for example by (1979) Biochemistry 18:5294-5299 such as Chirgwin guanidine thiocyanate extracting method) and by ThermoScript II (for example from Gibco/BRL, Bethesda, the Moloney MLV ThermoScript II that MD obtains, maybe can be from Seikagaku America, Inc., St.Petersburg, the AMV ThermoScript II that FL obtains) generation cDNA.

The synthetic oligonucleotide primer thing that [0128.0.0.0] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 1-5 is capable and/or the primer of 334-338 shown in capable to) can based on sequence as herein described (for example as Table I the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable or from as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence deutero-sequence) produce.

[0129.0.0.0] in addition, can therefrom can draw conservative region and can obtain degenerated primer conversely by carrying out the conservative region that multiple biology is identified in the protein sequence comparison with the employed polypeptide of the inventive method (polypeptide particularly of the present invention or be used for the sequence of the polypeptide of the inventive method).Polypeptide of the present invention or the shown in the drawings comparison of conservative region that is used for the polypeptide of the inventive method indicate.Conservative region is the zone of changes little in the aminoacid sequence of a specific position in several homologues of different sources.Table IV the 7th row, 1-5 is capable and/or 334-338 capable shown in consensus sequence from described comparison.

[0130.0.0.0] can use degenerated primer to have the new protein fragment of aforementioned activity (increasing as give each fine chemicals after improving expression or activity) or other the functional homologue that derives from the polypeptide of the present invention of other biological or be used for the polypeptide of the inventive method by pcr amplification then.

[0131.0.0.0] can use these fragments to separate complete genome sequence as hybridization probe then.As alternative, can pass through RACE-PCR (the terminal rapid amplifying of cDNA) method and separate 5 ' and the 3 ' sequence of omitting.Can use cDNA or genomic dna instead as template, use suitable Oligonucleolide primers according to the Standard PC R amplification technique nucleic acid molecule of the present invention that increases.Kuo Zeng nucleic acid molecule can be cloned into suitable carriers and be described its feature by the method for dna sequence analysis thus.Can be by the oligonucleotide of standard synthetic method (for example automatic dna synthesizer) generation corresponding to one of nucleic acid molecule that uses in the method.

The nucleic acid molecule that [0132.0.0.0] helps the inventive method can separate with the homology of nucleic acid molecule disclosed herein based on them, described separation uses this sequence or its part as hybridization probe, carries out according to the standard hybridization technique under stringent hybridization condition.In this article, for example can use and aforementioned nucleic acid molecule (particularly comprise in the inventive method nucleic acid molecule used therefor or code book and invent the nucleotide sequence of used proteinic nucleic acid molecule or the nucleotide sequence of nucleic acid molecule of the present invention) hybridize under stringent condition, and length is at least 15,20,25,30,35,40,50,60 or polynucleotide more, the isolated nucleic acid molecule of preferred at least 15,20 or 25 Nucleotide.Also can use and have 30,50,100,250 or the nucleic acid molecule of polynucleotide more.

[0133.0.0.0] term " homology " refers to that each nucleic acid molecule or coded protein are equivalent on function and/or structure.With the described nucleic acid molecule homology of preamble and for the nucleic acid molecule of the derivative of described nucleic acid molecule is the variant of described nucleic acid molecule that for example has the modification of identical biological function, particularly coding has the proteinic variant of identical or basic identical biological function.They can be naturally occurring variants, as derive from the sequence of other plant mutation or species or mutant.These mutant can be naturally occurring or be obtained by induced-mutation technique.Allelic variant can be the variant of naturally occurring allelic variant and synthetic generation or genetic modification.For example, can be by testing combining or identifying structural equivalents of described polypeptide and antibody based on computer forecast.Structural equivalents has similar immunological characteristic, for example contains similar epi-position.

[0134.0.0.0] " hybridization " refers to that these nucleic acid molecule hybridize under the conventional hybridization condition, preferably (Sambrook " Molecular Cloning for example under stringent condition; A LaboratoryManual ", second edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY (1989) or " Current Protocols in Molecular Biology ", JohnWiley ﹠amp; Sons, N.Y. (1989), the stringent condition described in the 6.3.1-6.3.6.) hybridization.

According to the present invention, the DNA of nucleic acid of the present invention and RNA molecule can be used as probe and use [0135.0.0.0].In addition, can be used as the template of identifying the function homologue and carry out Northern engram analysis and Southern engram analysis.The Nouthern engram analysis advantageously provides the further information about the expressed gene product: for example express generation of graphic, procedure of processing (as montage and add cap) etc.The S0uthern engram analysis provides about the chromosomal localization of the gene of code book invention nucleic acid molecule and the extraneous information of tissue.

The preferred limiting examples of [0136.0.0.0] stringent hybridization condition is under about 45 ℃, (=SSC) the middle hybridization of 6 * sodium chloride/sodium citrate, subsequently at 50 to 60 ℃, for example in 0.2 * SSC, 0.1%SDS, carry out the one or many washing step under 50 ℃, 55 ℃ or 60 ℃.The technician knows these hybridization conditions as the function of nucleic acid type and change, and (for example when having organic solvent) is relevant with the temperature and the concentration of damping fluid.Temperature under " standard hybridization conditions " is as the function of nucleic acid type and change, for example 0.1 *, 0.5 *, 1 *, 2 *, 3 *, 4 * or the aqueous buffer solution of 5 * SSC concentration in (pH7.2) between 42 ℃ and 58 ℃, preferably between 45 ℃ and 50 ℃.When having organic solvent (for example 50% methane amide) in the aforementioned damping fluid, the temperature under the standard conditions is about 40 ℃, 42 ℃ or 45 ℃.The hybridization conditions of DNA:DNA heterozygote for example is preferably 0.1 * SSC and 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ or 45 ℃, between preferred 30 ℃ and 45 ℃.The hybridization conditions of DNA:RNA heterozygote for example is preferably 0.1 * SSC and 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃ or 55 ℃, between preferred 45 ℃ and 55 ℃.The hybridization temperature that preamble is mentioned with for example when the no methane amide about 100bp of length (=base pair) and G+C content be that 50% nucleic acid is determined.By means of textbook or following textbook as the aforementioned, the technician understands how to determine required hybridization conditions: Sambrook etc., " Molecular Cloning ", Cold Spring HarborLaboratory, 1989; Hames and Higgins compiles 1985, " Nucleic AcidsHybridization:A Practical Approach ", IRL Press at Oxford UniversityPress, Oxford; Brown compiles 1991, " Essential Molecular Biology:A PracticalApproach ", IRL Press at Oxford University Press, Oxford.

Other examples of one of [0137.0.0.0] these stringent hybridization conditions are at 65 ℃, hybridize among 4 * SSC, wash one hour in 0.1 * SSC at 65 ℃ subsequently.Representative stringent hybridization condition in addition be in 50% methane amide, 4 * SSC under 42 ℃.In addition, the condition of washing step can be selected in the condition and range that is limited by low stringency condition (about 2 * SSC, 50 ℃) and high stringent condition (about 0.2 * SSC, 50 ℃, preferred 65 ℃) (20 * SSC:0.3M Trisodium Citrate, 3M sodium-chlor, pH7.0).In addition, the temperature of washing step can be brought up to about 65 ℃ of high stringent condition from the room temperature (about 22 ℃) of low stringency condition.Two parameters of salt concn and temperature can change simultaneously, or in two parameters one keeps constant and has only another variation.Also can in hybridization, use denaturing agent, for example methane amide or SDS.When having 50% methane amide, preferably hybridize at 42 ℃.Correlative factor such as i) handle length, ii) the salt condition, iii) the washing composition condition, iv) competitor dna, v) temperature and vi) the selection of probe can make up one by one, so this paper can not mention all possibilities.

Therefore, in preferred embodiments, (Roth is Karlsruhe) 68 ℃ of prehybridizations 2 hours for Northern blotting membrane and Rothi-Hybri-Quick damping fluid.Spend the night with radiolabeled probe hybridization at 68 ℃.Carry out washing step at 68 ℃ with 1 * SSC subsequently.For the Southern engram analysis, (Roth is Karlsruhe) 68 ℃ of prehybridizations 2 hours for film and Rothi-Hybri-Quick damping fluid.Cross liquid at 68 ℃ with radiolabeled probe hybridization.Abandon hybridization buffer subsequently, use 2 * SSC, 0.1%SDS to wash filter membrane in short-term.Add new 2 * SSC, 0.1%SDS damping fluid after abandoning lavation buffer solution, and hatched 15 minutes at 68 ℃.This washing step carries out twice, uses 1 * SSC, 0.1%SDS to carry out 10 minutes extra washing steps at 68 ℃ subsequently.

Some other example of [0138.0.0.0] DNA hybridization (Southern engram analysis) and washing step is as follows:

(1) hybridization conditions can be selected from for example following condition:

a)4×SSC、65℃，

b)6×SSC、45℃，

C) the fragmentation milt DNA of 6 * SSC, 100mg/ml sex change, 68 ℃,

D) salmon sperm DNA of 6 * SSC, 0.5%SDS, 100mg/ml sex change, 68 ℃,

E) the fragmentation salmon sperm DNA of 6 * SSC, 0.5%SDS, 100mg/ml sex change, 50% methane amide, 42 ℃,

F) 50% methane amide, 4 * SSC, 42 ℃,

G) 50% (volume/volume) methane amide, 0.1% bovine serum albumin, 0.1% phenanthrene can, 0.1% polyvinylpyrrolidone, 50mM sodium phosphate buffer (pH6.5), 750mM sodium-chlor, 75mM Trisodium Citrate, 42 ℃,

H) 2 * or 4 * SSC, 50 ℃ (low stringency condition), or

I) 30 to 40% methane amides, 2 * or 4 * SSC, 42 ℃ (low stringency condition).

(2) wash conditions can be selected from for example following condition:

A) 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1%SDS, 50 ℃,

b)0.1×SSC、65℃，

c)0.1×SSC、0.5％SDS、68℃，

D) 0.1 * SSC, 0.5%SDS, 50% methane amide, 42 ℃,

e)0.2×SSC、0.1％SDS、42℃，

F) 2 * SSC, 65 ℃ (low stringency condition).

[0139.0.0.0] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence, preferred Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence hybridization, and coding is expressed and is had the active peptide of the methionine(Met) of increasing.

[0140.0.0.0] in addition, some application can only be carried out under low stringent hybridization condition, and for the not influence of specificity of hybridization.For example, can carry out the Southern engram analysis of total DNA as probe with nucleic acid molecule of the present invention, and (in 2 * SSPE0,1%SDS under 55 ℃) washing under low stringency condition.Hybridization analysis can disclose the simple mode (activity that for example has each fine chemicals of increase as herein described) that the gene that has only used polypeptide in code book invention polypeptide or the inventive method just has.Another example of the low stringent hybridization condition of this class is 4 * SSC, 50 ℃ or use 30% to 40% methane amide 42 ℃ of hybridization.These molecules contain fragment, analogue or the derivative of the polypeptide that uses in polypeptide of the present invention or the inventive method, and by for example alone or in combination amino acid and/or nucleotide deletion, insertion, substitute, interpolation and/or reorganization or any other modifications known in the art are different from aforementioned aminoacid sequence or its coding nucleotide sequence.Yet, preferably use high stringent hybridization condition.

[0141.0.0.0] should be advantageously with at least 5,10,15,20,25,30,35 or 40bp, advantageously at least 50,60,70 or 80bp, preferred at least 90,100 or the fragment of 110bp hybridize.Most preferably at least 15,20,25 or the fragment of 30bp.Same preferred and the length fragment of 100bp or 200bp, extremely preferred 400bp are at least at least hybridized.In particularly preferred embodiments, should hybridize with complete nucleotide sequence subject to the foregoing.

[0142.0.0.0] term " fragment ", " sequence fragment " or " sequence part " refer to the truncated sequence of described original series.The length of truncated sequence (nucleic acid or protein sequence) can generally greatly be enclosed variation; Minimum length is for enough providing at least one suitable with described original series function and/or activity, or with nucleic acid molecule of the present invention or the inventive method or process of the present invention in the sequence of the nucleic acid molecule hybridize under stringent condition that uses, and maximum length is inessential.In some applications, maximum length is not usually significantly greater than activity and/or the required length of function that the original series that needs is provided.

[0143.0.0.0] usually, brachymemma length amino acid sequence scope from about 5 to about 310 amino acid.Yet more generally, sequence length is about 250 amino acid to the maximum, preferably is about 200 or about 100 amino acid to the maximum.Usually need to select at least about 10,12 or 15 amino acid, the most about 20 or 25 amino acid whose sequences.

[0144.0.0.0] term " epi-position " relates to the immune response site of antigen internal specific, is also referred to as antigenic determinant.These epi-positions can be monomeric linear array in the polymer composition (as the amino acid in the protein), perhaps form or comprise more complicated secondary or tertiary structure by more complicated secondary or tertiary structure.The technician will appreciate that immunogen (promptly can excite the material of immunne response) is an antigen; Yet some antigens such as haptens are not immunogens, but can be by producing immunogenicity with the carrier molecule coupling.Term " antigen " comprises and can produce antibody and/or the antibody material at its specific immune reaction at it.

[0145.0.0.0] the present invention relates to the epi-position of polypeptide in one embodiment, and described polypeptide is for polypeptide of the present invention or the employed polypeptide of the inventive method and give aforementioned activity, preferably gives the raising of each fine chemicals.

[0146.0.0.0] term " one or several amino acid " relates at least one amino acid, but is no more than the amino acid number that can cause homology to be lower than 50% identity.Identity preferably is higher than 70% or 80%, and more preferably 85%, 90%, 91%, 92%, 93%, 94% or 95%, even more preferably 96%, 97%, 98% or 99% identity.

[0147.0.0.0] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in one of nucleotide sequence, preferred Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.0.0] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, nucleotide sequence shown in the capable and/or 334-338 of 1-5 is capable, preferred Table I B the 5th or 7 row, nucleotide sequence or its funtion part homology were at least about 30% shown in the capable and/or 334-338 of 1-5 was capable, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases fine chemicals.

[0149.0.0.0] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, one of nucleotide sequence shown in the capable and/or 334-338 of 1-5 is capable, preferred Table I B the 5th or 7 row, one of nucleotide sequence shown in the capable and/or 334-338 of 1-5 is capable or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (as giving each fine chemicals increase) also as Table II the 5th or 7 row, during the capable and/or 334-338 of 1-5 is capable, preferred Table II B the 5th or 7 row, protein shown in the capable and/or 334-338 of 1-5 is capable.

[0149.1.0.0] randomly, with Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in one of nucleotide sequence, preferred Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd row, 1-5 is capable and/or 334-338 capable in, preferred Table II B the 3rd row, 1-5 is capable and/or the protein of 334-338 shown in capable is known activity or is used for these protein of note.

[0150.0.0.0] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor can only contain Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in one of sequence, preferred Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving methionine(Met) when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in one of sequence sense strand, Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of nucleotide sequence of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 1-5 is capable and/or 334-338 capable shown in the right PCR of primer will produce as Table I the 5th or 7 be listed as, 1-5 is capable and/or 334-338 capable shown in the fragment of polynucleotide sequence.Preferably Table II B the 7th is listed as, 1-5 is capable and/or 334-338 is capable.

[0151.0.0.0] primer sets is interchangeable.Those skilled in the art understand the required product of the described primer generation of combination in for example full-length clone or local sequence.Based on sequence of nucleic acid molecules of the present invention or the employed probe of the inventive method can be used to detect the transcript or the genome sequence of the identical or homologous protein of coding.Probe can also contain the labelling groups that connects thereon, and for example labelling groups can be radio isotope, fluorescent chemicals, enzyme or enzyme cofactor.The part that these probes can be used as genome marker test kit is used to identify the cell of expressing polypeptide of the present invention or polypeptide that the inventive method is used, as by measuring the level of coding nucleic acid molecule in the cell sample, whether the genomic gene that for example detects the mRNA level or determine to contain polynucleotide sequence of the present invention or polynucleotide sequence that the inventive method is used suddenlys change or lacks.

[0152.0.0.0] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coded polypeptide or its part, described polypeptide or its part comprise with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising methionine(Met) as be shown in the examples.

[0153.0.0.0] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in protein or its part have Table II the 3rd row for example, 1-5 is capable and/or 334-338 capable shown in the activity of polypeptide.

[0154.0.0.0] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.0] preferably has the activity of above stated specification by nucleic acid molecule of the present invention or the preferred biologically active of the inventive method nucleic acid molecule used therefor encoded protein matter part, and for example giving each fine chemicals after improving activity increases.

[0156.0.0.0] term mentioned in this article " biologically-active moiety " is intended to comprise the part of giving each fine chemicals increase, thereby or have immunocompetence and specificity in conjunction with polypeptide of the present invention or be used to produce the part that the antibody of the polypeptide that the inventive method is used of each fine chemicals combines, for example structural domain/motif.

[0157.0.0.0] the present invention relate in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase), for example comprise as Table IV the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in consensus sequence polypeptide or as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment as Table IV the 5th or 7 row, 1-5 is capable and/or the consensus sequence of 334-338 shown in capable or as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as Table IV the 5th or 7 row, 1-5 is capable and/or the consensus sequence of 334-338 shown in capable or as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the basic homology of aminoacid sequence of consensus sequence of polypeptide or its function homologue.Yet, in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable, does not preferably comprise as Table I A the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable.Preferably, nucleic acid molecule of the present invention is Table I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the function homologue of nucleic acid molecule or identical with it.

[0158.0.0.0] those of skill in the art will recognize that to have the dna sequence polymorphism that causes that aminoacid sequence changes in the population in addition.Code book invention polypeptide or be used for the polypeptide of the inventive method or contain nucleic acid molecule of the present invention or these genetic polymorphisms of the gene of the inventive method nucleic acid molecule used therefor can exist owing to natural variation in the individuality of population.

[0159.0.0.0] term used herein " gene " and " recombination " refer to the opening code-reading frame of the polypeptide that contains code book invention polypeptide or be used for the inventive method or contain nucleic acid molecule of the present invention or the nucleic acid molecule of the inventive method nucleic acid molecule used therefor or code book polypeptide that inventive method is used, preferably from crop plants or from the microorganism that can be used for producing each fine chemicals, especially for producing each fine chemicals.These natural variations generally cause the change of 1-5% in the nucleotide sequence of gene.At code book invention polypeptide or be used for the polypeptide of the inventive method or contain nucleic acid molecule of the present invention or the gene of the inventive method nucleic acid molecule used therefor, cause and do not change the arbitrary and whole of described functional activity by natural variation, such nucleotide diversity and the amino acid polymorphism that causes thereof are intended to be contained in the scope of the invention.

[0160.0.0.0] can be based on the homology of they and this nucleic acid molecule corresponding to the nucleic acid molecule (also can be cDNA) of the natural variant homologue of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor, use nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or its part as hybridization probe, under stringent hybridization condition, separate according to the standard hybridization technique.

[0161.0.0.0] therefore, in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor length are at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.0] term " hybridize under stringent condition " defines as preamble.In one embodiment, term " hybridize under stringent condition " is intended to describe hybridization and the wash conditions that at least 30%, 40%, the 50% or 65% identical general still maintenance of nucleotide sequence is each other hybridized mutually.Preferably, this condition make at least about 70%, more preferably at least about 75% or 80% in addition more preferably at least about 85%, 90% 95% or the sequence of higher identity generally still keep hybridization each other.

[0163.0.0.0] preferably, under stringent condition with Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the nucleic acid molecule of the present invention of sequence hybridization or the inventive method nucleic acid molecule used therefor corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.0] technician will also be understood that, the naturally occurring variant of polypeptide of the present invention in may reside in population or nucleic acid molecule and polypeptide that the inventive method is used or sequence of nucleic acid molecules, can in the nucleotide sequence of the nucleic acid molecule of code book invention polypeptide or polypeptide that the inventive method is used, introduce by sudden change and change, thereby cause that coded described amino acid sequence of polypeptide changes, and do not change polypeptide Functional Capability (preferably not reducing described activity).

[0165.0.0.0] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.0] " nonessential " amino-acid residue is can be changed in wild-type sequence and do not change the residue of this polypeptide active, and " essential " amino-acid residue is that aforementioned activity (for example causing after improving polypeptide active that each fine chemicals improves in the biology) is needed.Yet other amino-acid residues (for example not conservative or only semiconservative amino-acid residue in having described active structures territory) may not be active essential, therefore might be adapted at not changing under the described active condition changing.

[0167.0.0.0] it be known to those skilled in the art that it may be different that interbiotic codon is selected in addition.Therefore, the codon that may need to make the codon of nucleic acid molecule of the present invention to select to be adapted to express the biology of polynucleotide or polypeptide is selected.

[0168.0.0.0] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or 334-338 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or the aminoacid sequence of 334-338 shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, sequence shown in the capable and/or 334-338 of 1-5 is capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, one of sequence shown in the capable and/or 334-338 of 1-5 is capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, sequence shown in the capable and/or 334-338 of 1-5 is capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, sequence shown in the capable and/or 334-338 of 1-5 is capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.0] is for determining the percent homology (=identity) of two aminoacid sequences or two nucleic acid molecule, a sequence is write on another below, so that best relatively (for example the best for generation protein or nucleic acid and another protein or nucleic acid compares, and can insert breach in the sequence of protein or nucleic acid).

[0170.0.0.0] compares amino-acid residue on corresponding amino acid position or the nucleotide position or nucleic acid molecule then.If the position in sequence by with another sequence corresponding position on identical amino-acid residue or nucleic acid molecule when occupying, two molecules are homologous (being amino acid used herein or nucleic acid " homology " " identity " corresponding to amino acid or nucleic acid) on this position.Percent homology between two sequences is the function (being number/total number of positions * 100 of % homology=same position) of the same position number shared between sequence.Therefore term " homology " and " identity " are interpreted as synonym.

[0171.0.0.0] developed some kinds of computer software program in order to determine two or more amino acid or two or more homology of nucleotide sequence per-cent (=identity).Can use for example homology of the two or more sequences of fasta computed in software, fasta now has been applied to fasta 3 versions (W.R.Pearson and D.J.Lipman (1988), Improved Tools for BiologicalSequence Comparison.PNAS 85:2444-2448; W.R.Pearson (1990) Rapidand Sensitive Sequence Comparison with FASTP and FASTA, Methods inEnzymology 183:63-98; W.R.Pearson and D.J.Lipman (1988) ImprovedTools for Biological Sequence Comparison.PNAS 85:2444-2448; W.R.Pearson (1990); Rapid and Sensitive Sequence Comparison with FASTPand FASTAMethods in Enzymology 183:63-98).Another program that can be used for calculating different sequence homologies is the standard blast program that comprises in the Biomax pedant software (Biomax, Munich, Germany).Because blast does not always comprise the complete sequence of object and inquiry, may cause the suboptimum result sometimes.However, because this program is very effective, can use it for the comparison of a large amount of sequences.More generally use following the setting for such sequence :-p program name [character string];-d database [character string]; Acquiescence=nr;-i inquiry file [input file]; Acquiescence=stdin;-e expected value (E) [real number]; Acquiescence=10.0;-m compares Show Options: 0=pairing; 1=inquiry-, show identity than last zone; 2=inquiry-, do not show identity than last zone; 3=inquiry-, show identity than the screen literary composition form in last zone; 4=inquiry-, do not show identity than the screen literary composition form in last zone; 5=inquiry-than last zone, display consistency not, no unexpected end; 6=inquiry-than the screen literary composition form in last zone, display consistency not, no unexpected end; 7=XML Blast output; The 8=tabulation; The tabulation [integer] of 9 band comment lines; Acquiescence=0;-o

BLAST report output file [output file] is optional; Acquiescence=stdout;-F filters search sequence (blastn is DUST, and other are SEG) [character string]; Acquiescence=T;-G opening cost (acyclic homologically trioial default behavior) [integer]; Acquiescence=0;-E opening cost (acyclic homologically trioial default behavior) [integer]; Acquiescence=0;-X X breach comparison falling-threshold value (bit) (acyclic homologically trioial default behavior); Blastn 30, and megablast 20, and tblastx 0, other 15[integers]; Acquiescence=0;-l shows GI ' s[T/F in Presenting Bank]; Acquiescence=F;-q Nucleotide mispairing point penalty (only being used for blastn) [integer]; Acquiescence=-3; The prize of-r Nucleotide coupling divides (only being used for blastn) [integer]; Acquiescence=1;-v shows the database sequence number [integer] that the single file of (V) is described; Acquiescence=500;-b shows the database sequence number [integer] of (B) comparison; Acquiescence=250;-f extends hit threshold, if be 0 then acquiescence; Blastp 11, and blastn 0, and blastx 12, and tblastn 13; Tblastx 13, megablast 0[integer]; Acquiescence=0;-g implements breach comparison (tblastx is unavailable) [T/F]; Acquiescence=T; The genetic code [integer] that-Q inquiry is used; Acquiescence=1;-D DB genetic code (only be used for tblast[nx]) [integer]; Acquiescence=1; The treater number [integer] that-a uses; Acquiescence=1;-O SeqAlign file [output file] is optional;-J Believe the query defline[T/F]; Acquiescence=F;-Metzler matrix [character string]; Acquiescence=BLOSUM62;-W word length is if be 0 then acquiescence (blastn 11, megablast28, other are 3 years old) [integer]; Acquiescence=0;-z database useful length (using 0) [real number] for true length; Acquiescence=0;-K keeps district's hits (acquiescence is closed, if use, recommendation is 100) [integer]; Acquiescence=0; Multiple the hitting of-P is 0, singly hits the integer into 1[]; Acquiescence=0;-Y search space useful length (using 0) [real number] for true length; Acquiescence=0;-S at the inquiry chain of database search (be used for blast[nx] and tblastx); 3 are both, and 1 is the top, and 2 is below [integer]; Acquiescence=3;-T produces HTML output [T/F]; Acquiescence=F; The restricted search database of-I is to list GI ' s[character string] optional; It is optional that-U uses small letter to filter [T/F] to the FASTA sequence; Acquiescence=F; The non-breach of-y X extends and reduces threshold ratio paricular value (0.0 calls default behavior); Blastn 20, and megablast 10, every other 7[real number]; Acquiescence=0.0; The final breach comparison of-Z X reduces threshold ratio paricular value (0.0 calls default behavior); Blastn/megablast 50, tblastx0, every other 25[integer]; Acquiescence=0;-R PSI-TBLASTN check point file [input] is chosen wantonly;-n MegaBlast searches for [T/F]; Acquiescence=F;-L search sequence location [character string] is optional; The multiple window size that hits of-A, if be 0 the acquiescence (blastn/megablast 0, every other 40[integer]; Acquiescence=0;-w frameshit point penalty (blastx is the OOF algorithm) [integer]; Acquiescence=0; Allow to connect the maximum intron length (0 interrupts connecting) [integer] of HSPs among the-t tblastn; Acquiescence=0.

[0172.0.0.0] uses the algorithm of Needleman and Wunsch or Smith and Waterman to obtain high quality results.Therefore be preferably based on the program of described algorithm.Advantageously, can service routine PleUp (J.Mol.Evolution., 25,351-360,1987, Higgins etc., CABIOS, 51989:151-153) or preferred program Gap and BestFit obtain sequence relatively, they are respectively based on the algorithm (J.Mol.Biol.48 of Needleman and Wunsh; 443-453 (1970)) and the algorithm (Adv.Appl.Math.2 of Smith and Waterman; 482-489 (1981)).Two programs all be the GCG software package (Genetics Computer Group, 575Science Drive, Madison, Wisconsin, USA 53711 (1991); Altschul etc. (1997) Nucleic Acids Res.25:3389 and following or the like) a part.Therefore preferably on the complete sequence scope, use the Gap program to finish calculating to determine sequence homology per-cent.Nucleotide sequence relatively used following standard adjustment: breach weight: 50, length weight: 3, average coupling: 10.000, average mispairing: 0.000.

[0173.0.0.0] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:1 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:1 sequence before use.

[0174.0.0.0] is in the current level of this area, homology between two polypeptide also is interpreted as referring to by by GAP (Wisconsin Package Version 10.0, University ofWisconsin, Genetics Computer Group (GCG), Madison, USA) amino acid sequence identity under the complete sequence length situation that relatively calculates of programmed algorithm is set to following parameter:

Breach weight: 8 length weights: 2

Average coupling: 2,912 average mispairing :-2,003

[0175.0.0.0] for example, the sequence that has 80% homology at protein level and SEQ ID NO:2 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ IDNO:2 sequence.

[0176.0.0.0] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in the capable and/or 334-338 of 1-5 was capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in the capable and/or 334-338 of 1-5 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in the capable and/or 334-338 of 1-5 is capable has essentially identical character and discerns.

[0177.0.0.0] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, 1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, the functional equivalent that nucleotide sequence obtained shown in the capable and/or 334-338 of 1-5 was capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in the capable and/or 334-338 of 1-5 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding has and is listed as Table II the 5th or 7,1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, the polypeptide of the essentially identical character of polypeptide shown in the capable and/or 334-338 of 1-5 is capable.

[0178.0.0.0] functional equivalent " essentially identical characteristic " at first is interpreted as functional equivalent and has activity mentioned above, for example, increase the amount of each fine chemicals improving described functional equivalent when biology is as albumen quantity, activity or function in microorganism, plant or plant or animal tissues, plant or zooblast or its part.

[0179.0.0.0] can by to the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in) in introduce replacement, interpolation or the disappearance of one or more Nucleotide, and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th is listed as, 1-5 is capable and/or 334-338 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 introduces sudden change in the encoding sequence of sequence shown in capable.

[0180.0.0.0] preferably carries out conservative amino acid and replaces at the non-essential amino acid residue place of one or more predictions." conservative amino acid replacement " is the replacement with the amino-acid residue substituted amino acid residue with similar side chain.This area has defined the amino-acid residue family with similar side chain.These families comprise amino acid with basic side chain (Methionin for example, arginine, Histidine), amino acid (aspartic acid for example with acid side-chain, L-glutamic acid), amino acid (glycine for example with uncharged polar side chain, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), amino acid (L-Ala for example with non-polar sidechain, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), amino acid (Threonine for example with β branched building block, Xie Ansuan, Isoleucine) and have the amino acid (tyrosine for example of aromatic side chain, phenylalanine, tryptophane, Histidine).

[0181.0.0.0] therefore, polypeptide of the present invention or the inventive method use the non-essential amino acid residue predicted in the polypeptide preferably by the aminoacid replacement from same side chain family.Alternatively, in another embodiment, can can keep even have the mutant of the aforementioned activity (for example giving each fine chemicals content improves) of raising at screening active ingredients gained mutant described herein with evaluation as in the encoding sequence of all or part of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used, introducing sudden change at random by saturation mutagenesis.

After [0182.0.0.0] carries out mutagenesis to one of sequence shown in this paper, can recombinant expressed encoded protein matter also can use assay method as described herein (seeing embodiment) to measure activity of proteins.

[0183.0.0.0] found nucleic acid molecule that the inventive method is used and the highest homology of following data base entries by the Gap search.

[0184.0.0.0] is employed to have as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in the capable and/or 334-338 of 1-5 is capable, perhaps come Table II the 5th or 7 row freely, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, the homologous compound of the nucleotide sequence of sequence shown in the capable and/or 334-338 of 1-5 is capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.0.0] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, 1-5 is capable and/or the sequence of 334-338 shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, 1-5 is capable and/or the arbitrary sequence of 334-338 shown in capable in other nucleotide sequence of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, 1-5 is capable and/or the sequence of 334-338 shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.0.0] also preferred the inventive method comprise as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or 334-338 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or 334-338 capable shown in sequence identical.

[0187.0.0.0] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or 334-338 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or 334-338 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.0.0] still has the polypeptide basic enzyme activity of the present invention (being its active basic reduction that do not have) of giving each fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, polypeptide active shown in the capable and/or 334-338 of 1-5 is capable, preferably with Table II the 3rd and 5 row, shown in the capable and/or 334-338 of 1-5 is capable and the sequence of expressing under the same conditions compare and do not reduce substantially.

In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, 1-5 is capable and/or 334-338 capable shown in sequence or by its homologue of forming.

[0189.0.0.0] as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the homologous compound of sequence, or deutero-as Table II the 5th or 7 row, 1-5 is capable and/or the homologue of the sequence of 334-338 shown in capable also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.0] in another embodiment, method of the present invention may further comprise the steps:

(a) biological or its part of polypeptide of the present invention is expressed in selection;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with institute mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutagenesis biology or its part of expression level with selected biology or its part (a);

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or each fine chemicals of bonded of selected mutation biology or the generation of its part.

Biological or its part of [0191.0.0.0] and described contrast or selection is compared, should biology or its part produces free and/or each fine chemicals level of bonded improves according to the inventive method described herein.

[0191.1.0.0] compares with biological or its part of described contrast or selection in one embodiment, according to the inventive method described herein should biology or its part each fine chemicals level of producing protein bound improve.

[0192.0.0.0] advantageously, selected biology is the biology of the mutagenesis according to the present invention.According to the present invention, mutagenesis is any variation of genetic information in the biological gene group, promptly is not because any structure of the biotinylated nucleic acid that normal separation or genetic recombination process cause (preferred DNA) or the variation of composition.These sudden changes can spontaneously occur, and perhaps can induce by mutagenic compound hereinafter described.These variations can be induced or selective induction at random.In both cases, biological heredity information is all modified.General this can cause in the cell or the gene product of biological interior genes involved activity improves.

For the situation of special mutagenesis or so-called site-directed mutagenesis, unique gene is suddenlyd change [0193.0.0.0], so its activity inhibited, reduction or raising (the preferred raising) active and/or coded gene product.In random mutagenesis, one or more genes are arbitrarily suddenlyd change, and its activity inhibited, reduction or raising (the preferred raising) active and/or its gene product.

[0194.0.0.0] can transform such colony with DNA construct with regard to mutagenesis mcroorganism colony, described DNA construct can be used for activating gene as much as possible in the biology, preferred full gene.For example construct can contain near strong promoter or one or more enhanser of the gene can its integration site of transcription activating.Adopt this method, by random integration activation construct, can the nearly all statistically biological gene of mutagenesis (for example activation).The technician can identify that gene of the present invention is activated and and then cause that each fine chemicals of raising that needs produces the mutagenic fungi system that increases then.

[0195.0.0.0] also can activate gene of the present invention by mutagenesis regulatory region or coding region.For the situation of random mutagenesis, handle large number of biological with mutagenic compound.The amount of described reagent and the intensity of processing are according to each transgenation mode is once selected.Random mutagenesis methods and reagent separately are that the technician knows.These methods are disclosed in for example A.M.van Harten[(1998), " Mutation breeding:theory and practical applications ", Cambridge University Press, Cambridge, UK], E Friedberg, G Walker, W Siede[(1995), " DNA Repair and Mutagenesis ", BlackwellPublishing], or K.Sankaranarayanan, J.M.Gentile, L.R.Ferguson[(2000) " Protocols in Mutagenesis ", Elsevier Health Sciences].It is very low that the technician understands in the biomass cells spontaneous mutation rate, and number of chemical, physics or biological reagent can use for mutagenesis is biological.These reagent are called mutagen or mutagenic compound.As indicated above, three kinds of different types of mutagens (chemistry, physics or biological reagent) are available.

[0196.0.0.0] chemical mutagen has dissimilar, can distinguish by its binding mode.Base analogue for example is as 5-bromouracil, 2-aminopurine.Other chemical mutagens and DNA interact, as sulfuric acid, nitrous acid, azanol, or other alkylating agent, as single functional agent (as ethyl methane sulfonate, methyl-sulfate, methyl mesylate), difunctional dose (as Dichloroethyl thioether, mitomycin, nitrosoguanidine-dialkyl group nitrous acid, N-nitrosoguanidine derivative, N-alkyl-N-nitro-N-nitrosoguanidine), chimeric dyestuff, as acridine, the pyridine of bromine second.

[0197.0.0.0] physical mutagen is ionizing irradiation (X ray), UV irradiation for example.Can use dissimilar irradiations, they are strong mutagens.Can distinguish the irradiation of two main types: a) unionized irradiation (as UV light) or ionizing irradiation (as X ray).The biological induced-mutation factor is transposable element for example, as IS element (as IS100), transposon (as Tn5, Tn10, Tn916 or Tn1000) or phage, as Mu ^Amplac, P1, T5, lambda particles phage etc.The method of microorganism that this phage DNA introducing is suitable is known by the technician (consults " Microbiology ", the third edition, Davis volume, B.D., Dulbecco, R., Eisen, H.N. and Ginsberg, H.S., Harper InternationalEdition, 1980).The common method of transposon mutagenesis be gene or near (for example promotor or stop subarea) insert transposable element, thereby cause the forfeiture of gene function.The method that transposon is positioned in the biological gene group is well known to those skilled in the art.

[0198.0.0.0] preferably uses chemistry or biological method mutagenesis biology.Preferred chemical process is with N-methyl-N-nitro-nitroso-guanidine mutagenesis.

[0199.0.0.0] other biological method by Spee etc. (Nucleic Acids Research, volume 21, No.3,1993:777-778) open.Spee etc. have instructed the PCR method of using the dITP random mutagenesis.This method that Spee etc. describe is by (Protein Expr.Purif., 5,1994:270-277) further improvement such as Rellos.Stemmer (Proc.Natl.Acad.Sci.USA, 1994:10747-10751) described the use extracorporeal recombination and carried out molecule mutagenesis by volume 91.(NatureBiotechnology volume 14 1996:458-467) has been described combination PCR and recombination method to improve the enzymic activity of esterase to right-nitrobenzyl ester to Moore etc.(volume 57 has been described another approach of mutagenesis enzyme in 1996:375-385) to Greener etc. at Methods in Molecular Biology.Use specificity coli strain XL1-Red such as Greener produce the intestinal bacteria mutant of the antibiotics resistance with raising.

[0200.0.0.0] in one embodiment, the nucleic acid molecule that protein or this paper of the present invention characterized derives from eucaryon or prokaryotic organism, as non-human animal, plant, microorganism (as fungi, yeast, algae, diatom or bacterium).Advantageously the inventive method nucleic acid molecule used therefor derives from yeast, Saccharomycetaceae for example, be specially Saccharomycodes or such as mycocandida, Hansenula anomala genus, Pichia, inferior sieve yeast belong, red yeast belong or Schizosaccharomyces yeast, and particularly advantageously from yeast saccharomyces cerevisiae.

[0201.0.0.0] in one embodiment, advantageously the inventive method nucleic acid molecule used therefor derives from bacterium, Proteobacteria for example, γ-distortion Gammaproteobacteria particularly, be more preferably from the enterobacteria order, for example enterobacteriaceae, particularly Escherichia, salmonella, Klebsiella are advantageously from the e. coli k12 species.

[0202.0.0.0] if select plant as the donor biology in the methods of the invention, can there be any phylogenetic relation in this plant with recipient plant in principle.Donor and recipient plant can belong to same section, genus, kind, mutation or strain system, and the homology of nucleic acid to be integrated and recipient plant genome corresponding section is improved.This can also be applied to the microorganism as donor and receptor biological similarly.Can also advantageously use from the unusual nucleic acid molecule of different plant species, because the susceptibility that they may show endogenous regulation mechanism reduces, and sequence may be by endogenous reticent mechanism identification.

[0203.0.0.0] therefore, the application's a embodiment relates to be used in the methods of the invention from nucleic acid molecule of plant or their homologue, described plant is crop plants for example, as colea; Soybean; Sunflower Receptacle; Linseed oil or corn.

[0204.0.0.0] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

A) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or polypeptide or its segmental nucleic acid molecule of 334-338 shown in capable, described nucleic acid molecule is given each fine chemicals quantity in biological or its part increase;

B) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, 1-5 is capable and/or nucleic acid molecule or its segmental nucleic acid molecule of 334-338 shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

D) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

E) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

F) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

G) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

H) nucleic acid molecule, it comprises by using as Table III the 7th row, 1-5 is capable and/or the primer of 334-338 shown in capable or primer obtain amplification from cDNA library or genomic library nucleic acid molecule, and give the increase of each fine chemicals quantity in biology or its part;

I) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

J) encoded polypeptides contain just like Table IV the 7th row, 1-5 is capable and/or 334-338 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

K) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described peptide coding as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, 1-5 is capable and/or the structural domain of the polypeptide of 334-338 shown in capable; With

L) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7,1-5 is capable and/or 334-338 is capable, preferred Table I B the 7th row, nucleic acid molecule shown in the capable and/or 334-338 of 1-5 is capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table II B the 7th row, at least the 15nt of the nucleic acid molecule of polypeptide shown in the capable and/or 334-338 of 1-5 is capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby, the nucleic acid molecule of preferred (a) to (l) by one or more Nucleotide be different from as Table I A or I B the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable.In one embodiment, nucleic acid molecule not by Table I A or I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in and the sequence described form.In one embodiment, nucleic acid molecule and Table I A or I B the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II A or II B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide of sequence.In another embodiment, nucleic acid molecule of the present invention and Table I A or I B the 5th or 7 row, 1-5 is capable and/or 334-338 row shown in sequence at least 30%, 40%, 50% or 60% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as Table II A or II B the 5th or 7 row, 1-5 is capable and/or the peptide sequence of 334-338 shown in capable.Therefore, in one embodiment, nucleic acid molecule and Table I A or I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in nucleic acid molecule have at least one or more residues different.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or I B the 5th or 7 row, 1-5 is capable and/or the polypeptide of 334-338 shown in capable at least one or the different polypeptide of a plurality of amino acid.In another embodiment, Table I A or I B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in nucleic acid molecule do not encode Table II A or II B the 5th or 7 row, 1-5 is capable and/or the protein sequence of 334-338 shown in capable.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II A or II B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A or II B the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in protein sequence at least 30%, 40%, 50% or 60% identical and with Table I A or IIB the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.0] advantageously introduces the employed nucleotide sequence of present method in the nucleic acid construct (preferred expression box), and described nucleic acid construct might be expressed this nucleic acid molecule in biological (advantageously being plant or microorganism).

[0206.0.0.0] therefore the invention still further relates to the nucleic acid construct that contains the nucleic acid molecule of the present invention that is connected with one or more regulatory elements or semiotic function, the preferred expression construct.

[0207.0.0.0] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.0] says in principle, and nucleic acid construct can contain adjusting sequence as herein described and other sequences relevant with containing genetic expression.Therefore, nucleic acid construct of the present invention can be used as that expression cassette uses and thereby can directly use its introduced plant, perhaps they can be introduced carrier.Therefore, in one embodiment, nucleic acid construct is to contain microorganism promotor or microorganism terminator or both expression cassette.In another embodiment, expression cassette contain plant promoter or plant terminators or both.

[0209.0.0.0] therefore, in one embodiment, method of the present invention may further comprise the steps:

(a) introduce nucleic acid construct, described nucleic acid construct contains the nucleic acid molecule of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used or code book invention polypeptide or polypeptide that the inventive method is used; Or

(b) introduce the nucleic acid molecule that comprises the adjusting sequence or the factor, its expression has improved cell or expression biological or the middle nucleic acid molecule of the present invention of its part (preferred plant, vegetable cell or microorganism) or nucleic acid molecule that the inventive method is used or code book invention protein or proteinic nucleic acid molecule that the inventive method is used; With

(c) in cell or biology, express (a) or (b) in nucleic acid construct or the coded gene product of nucleic acid molecule mentioned.

Behind [0210.0.0.0] introducing and the express nucleic acid construct, advantageously cultivate and gather in the crops subsequently genetically modified organism or cell.Genetically modified organism or cell can be protokaryon or eukaryote, as microorganism, non-human animal and plant, and for example plant or zooblast, plant or animal tissues, preferred crop plants or its part.

[0211.0.0.0] introduces the nucleic acid molecule part of expression cassette (for example as) in the nucleic acid construct, advantageously in mode known to the skilled the encoding gene fragment increased and ligation.Preferably follow with Pfu archaeal dna polymerase or Pfu/Taq archaeal dna polymerase mixture and operate similarly.According to sequence selection primer to be amplified.Should select primer rightly so that amplified material comprises the mode of the encoding sequence from initial to terminator codon.After the amplification, analysing amplified rightly thing.For example, analysis can be considered quality and quantity, and carries out after separating by gel electrophoresis.Can follow standard method (for example Qiagen) purifying amplified material subsequently.The aliquots containig of purifying amplified material can be used for clone's step subsequently.The technician understands suitable cloning vector usually.

They specifically comprise the carrier that can duplicate [0212.0.0.0] in easy-to-handle cloning system (for example bacterium yeast or based on the system of insect cell (for example baculovirus expression)), promptly particularly guarantee in intestinal bacteria effective clone and carrier that may the stable conversion plant.The carrier that must mention is in particular the multiple binary that is suitable for the T-DNA mediated transformation and is total to the integrative vector system.These carrier systems are characterized by it usually and contain required vir gene of agrobacterium mediation converted and T-DNA border sequence at least.

[0213.0.0.0] generally speaking, carrier system preferably also contains other cis regulatory regions (as promotor and terminator) and/or in order to identify the selectable marker of suitable inverting biological.For integrative vector system altogether, vir gene and T-DNA sequence are positioned on the identical carrier, and binary vector is based at least two carriers, and one of them carries the vir gene, does not carry T-DNA, and second carried T-DNA, do not carry the vir gene.Based on this fact, last-mentioned carrier is less relatively, and easy handling also can duplicate in intestinal bacteria and Agrobacterium.These binary vectors comprise the carrier from pBIB-HYG, pPZP, pBecks, pGreen series.That preferably used according to the invention is Bin19, pBI101, pBinAR, pGPTV and pCAMBIA.Hellens etc., Trends in PlantScience (2000) 5,446-451 have provided the general introduction of binary vector and uses thereof.

[0214.0.0.0] at first uses restriction endonuclease with the carrier linearizing for the preparation carrier, uses enzyme modification then by rights.Subsequent purificn carrier, a part are used to clone step.In clone's step, use the carrier segments of amplified material that ligase enzyme is cut enzyme and purifying in case of necessity and same preparation to be cloned into together.In this article, specific nucleic acid construct or carrier or plasmid construction body can have one or more encoding gene fragments.Encoding gene fragment in these constructs preferably effectively is connected with the adjusting sequence.Regulate sequence and specifically comprise the plant sequence, as described above promotor and terminator.Can be under selection condition advantageously particularly stably breed construct and allogeneic dna sequence DNA can be transferred in plant or other microorganisms in intestinal bacteria and/or the agrobacterium tumefaciens (Argobacterium tumifaciens) in microorganism.According to a specific embodiment, construct is based on binary vector (binary vector general introduction: Hellens etc., 2000).Usually they contain protokaryon adjusting sequence (as replication orgin) and the selectable marker that is useful on propagation in microorganism (as intestinal bacteria and agrobacterium tumefaciens).Carrier can also contain to be useful on transfers to agrobatcerium T-DNA sequence in the Plant Genome with DNA, perhaps be used for transferring to other eucaryons adjusting sequences of other eukaryotic cells (as Saccharomycodes), perhaps be used for transferring to other protokaryons adjusting sequences of other prokaryotic cell prokaryocytes (as Corynebacterium or bacillus).In order to transform plant, advantageously comprised the right side boundary sequence of the total agrobatcerium T-DNA sequence that contains 25 base pairs of having an appointment.Plant conversion carrier construct of the present invention contains the T-DNA sequence in the right and left boundary zone usually, and the appropriate recognition site of locus specificity effect enzyme is contained in described zone, otherwise these enzymes are by some virogene codings.

[0215.0.0.0] suitable host living beings is that the technician is well-known.Favourable biology is that the application is mentioned above.They specifically comprise eukaryote or eubacterium, for example prokaryotic organism or archeobacteria.Favourable host living beings is to be selected from following microorganism: Actinomy cetaceae, Bacillaceae, Brevibacteriaceae, the rod Bacteriaceae, enterobacteriaceae, Gordon Salmonella section, micrococcaceae, mycobacteriaceae, Nocardiaceae, pseudomonadaceae, Rhizobiaceae, Streptomycetaceae, chaetomium section, the mould section of hairpin, Cryptococcaceae, Cunninghamellaceae, the dark born of the same parents section that obstructs, Moniliaceae, Mortierellaceae, Mucoraceae, pythiaceae, Saccharomycetaceae, Saprolegniaceae, fission yeast section, Sodariaceae section, Sporobolomycetaceae, Tuberculariaceae, Adelotheciaceae section, Dinophyceae, ox hair moss section and green branch algae guiding principle.Preferred unicellular microorganism is fungi, bacterium or protozoon for example, for example fungi such as Claviceps or Aspergillus, or gram-positive microorganism such as Bacillaceae, Corynebacterium, micrococcus sp, brevibacterium sp, Rhod, Nocardia, butter bacillus genus or genus arthrobacter, or Gram-negative bacteria such as Escherichia, Flavobacterium or salmonella, or yeast such as Rhodotorula, Hansenula anomala genus, Pichia, inferior sieve yeast belong, yeast belong, Schizosaccharomyces or mycocandida.

The host living beings that [0216.0.0.0] the inventive method particularly advantageous is selected is to be selected from the microorganism that comprises with subordinate and kind: Hansenula anomala, Candida utilis, ergot, Bacillus circulans, Bacillus subtilus, bacillus certain (Bacillus sp.), Brevibacterium albidum, Brevibacterium album, Brevibacterium cerinum, brevibacterium flavum, Brevibacterium glutamigenes, brevibacterium iodinum, Brevibacteriumketoglutamicum, brevibacterium lactofermentum, extension brevibacterium, Brevibacteriumroseum, Brevibacterium saccharolyticum, brevibacterium sp certain (Brevibacterium sp.), Corynebacterium acctoacidophlum, Corynebacterium acetoglutamicum, produce ammonia rod bacillus, Corynebacterium glutamicum (=micrococcus glutamicus), corynebacterium melassecola, Corynebacterium or intestinal bacteria, particularly e. coli k12 and described bacterial strain thereof.

[0217.0.0.0] be favourable preferably agrobacterium tumefaciens or plant host biology according to the present invention.Preferred plant is selected from following section: Aceraceae, Anacardiaceae, umbelliferae, composite family, umbelliferae, Betulaceae, Boraginaceae, brassicaceae, Bromelia family, Cactaceae, Caricaceae, Caryophyllaceae, Cannabaceae, convolvulaceae, Chenopodiaceae, Elaeangnaceae, Mang ox seedling section, Gramineae (Gramineae), Juglandaceae, Lauraceae, pulse family (Leguminosae), flax family (Linaceae), Curcurbitaceae, Cyperaceae, Euphorbiaceae, pulse family (Fabaceae), Malvaceae, Nymphaeceae, papaveracease, the Rosaceae, Salicaceae, Solanaceae, Palmae (Arecaceae), Iridaceae, Liliaceae, the orchid family, Gentianaceae, Labiatae, Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae, scrophulariaceae, Ericaceae, polygonaceae, Violaceae, rush family, Gramineae (Poaceae), perennial herb, fodder crop, vegetables and ornamental plant.

[0218.0.0.0] particularly preferably is the plant that is selected from following section: umbelliferae, composite family, brassicaceae, Curcurbitaceae, pulse family (Fabaceae), papaveracease, the Rosaceae, Solanaceae, Liliaceae or Gramineae (Poaceae).Particularly, particularly advantageous is crop plants.Therefore, preferred favourable plant belongs to peanut (peanut), rape (oilseed rape), rape (canola), Sunflower Receptacle (sunflower), safflower (safflower), olive (olive), sesame (sesame), fibert (hazelnut), apricot (almond), avocado (avocado), bay, pumpkin (pumpkin/squash), linseed oil (linseed), soybean (soya), pistachio (pistachio), Borrago officinalis (borage), corn (maize), wheat (wheat), rye (rye), oat (oats), jowar (sorghum) and millet (millet), triticale (triticale), rice (rice), barley (barley), cassava (cassava), potato (potato), sugar beet (sugarbeet), fodder beet (fodder beet), eggplant (egg plant) and perennial herb and forage plant, coconut oil (oilpalm), vegetables (brassicas, root vegetable, tuberous vegetable, beanpod class vegetables, the fruits vegetables, onion class vegetables, leafy vegetable and stem vegetables), buckwheat (buckwheat), jerusalem artichoke (Jerusalem artichoke), broad bean (broad bean), common vetch (vetches); root of Szemao crotalaria (lentil); alfalfa (alfalfa); string bean (dwarf bean); lupine (lupin); trifolium (clover) and alfalfa (lucerne).

[0219.0.0.0] by being used nucleic acid molecule of the present invention or the inventive method in the nucleic acid molecule introduced plant, and verified at first it to be transformed into intermediate host's (as bacterium or unicellular eukaryote cell) be favourable.Proved that transformed into escherichia coli is appropriate in this article, conversion can be carried out in a manner known way, for example the method by heat shock or electroporation.Therefore, can analyze the cloning efficiency of transformed into escherichia coli bacterium colony.This can be undertaken by PCR.Can be by confirming the identity and the integrity of plasmid construction body by the aliquots containig of bacterium colony being carried out colony number that described PCR defines.For this purpose, use universal primer usually from the carrier sequence, for example forward primer can be arranged in initial ATG upstream, reverse primer is arranged in the segmental terminator codon of encoding gene downstream.By the electrophoretic separation amplified material and assess its quality and quantity.

[0220.0.0.0] will (confirm randomly) that subsequently nucleic acid construct is used to transform plant or other hosts, as other eukaryotic cells or other prokaryotic cell prokaryocytes.At first need to obtain construct from middle host for this reason.For example, can from host bacterium, obtain construct (as plasmid) by being similar to the isolating method of traditional plasmid.

[0221.0.0.0] can also introduce the virus vector of modifying with nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used, as be used for baculovirus vector or plant viral vector in expressed in insect cells, as tobacco mosaic virus (TMV) or based on the carrier of potato virus X.Cause that protein comprises the infectious RNA inoculation tobacco of for example using from the cDNA copy in-vitro transcription of recombinant virus genomes from the approach of institute's modification virus genomic expression, described viral genome comprises nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used.The wound of another approach utilization inoculation agrobacterium tumefaciens transforms whole plants, and described agrobacterium tumefaciens contains the cDNA copy of the just RNA viruses of recombinating.The different carriers and the virus that are used for different targets (as producing plant) expression are known for the technician.

The big metering method of [0222.0.0.0] known conversion plant.According to the present invention, the stable integration allogeneic dna sequence DNA is favourable in Plant Genome, has proved that therefore the conversion of T-DNA-mediation is suitable especially.This purpose at first needs to transform suitable media, particularly Agrobacterium with encoding gene fragment that contains nucleic acid molecule of the present invention or corresponding plasmid construction body.This can carry out in a manner known way.For example, method that can be by electroporation or heat shock will transform in the competence Agrobacterium according to nucleic acid construct described of the present invention that above-detailed produced or described expression construct or described plasmid construction body.Say that in principle necessary difference formation is total to the one side of integrative vector and transforms on the other hand with binary vector.In the first string, the construct that contains encoding gene fragment of the present invention or nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor does not have the T-DNA sequence, and by in Agrobacterium, forming integrative vector or construct altogether with the construct allos reorganization that has T-DNA.T-DNA is present in the Agrobacterium with the form of Ti or Ri plasmid, and foreign DNA has replaced oncogene suitably in described plasmid.If the use binary vector can or directly shift they are transferred in the Agrobacterium by the bacterium combination.These Agrobacteriums have advantageously contained the carrier (being called auxiliary Ti (Ri) plasmid now) that carries the vir gene.

[0223.0.0.0] will be if plant or vegetable cell will transform with T-DNA, can also use one or more marks with nucleic acid construct or carrier of the present invention expediently, can separate or select the biology that transforms by mark, as Agrobacterium or plant transformed cell.These marker gene make can be by the successful conversion of a series of different methods evaluations nucleic acid molecule of the present invention, described method for example by the light visual identification in fluorescence, the luminous or human eye visible wavelength region, by to weedicide or antibiotic resistance, by be known as nutrition mark (nutrient defect type mark) or anti-nutrition mark, by the enzyme analysis or pass through plant hormone.The example of these marks that can mention is GFP (=green fluorescent protein), the luciferin/luciferase system, beta-galactosidase enzymes and coloured substrate (as X-Gal) thereof, Herbicid resistant is (as imidazolone, glyphosate, phosphinothricin or sulfonylurea), antibiotics resistance is (as bleomycin, Totomycin, Streptomycin sulphate, kantlex, tsiklomitsin, paraxin, penbritin, gentamicin, Geneticin (G418), spectinomycin or blasticidin) etc., nutrition mark (as utilizing seminose or wood sugar) or anti-nutrition mark (as 2-deoxyglucose resistance).Only listed the possible mark of sub-fraction herein.The technician is very familiar to these marks.Depend on biology and system of selection, different being labeled as is preferred.

[0224.0.0.0] generally needs the one or both sides of T-DNA at plant nucleic acid construct encoding gene fragment flank.This is particularly useful when using agrobacterium tumefaciens or Agrobacterium rhizogenes (Agrobacterium rhizogenes) to transform.Preferable methods of the present invention is the conversion by agrobacterium tumefaciens.Yet biological projectile method also can be advantageously used in the sequence of introducing in the inventive method, can also use the PEG method to introduce.Can cultivate the Agrobacterium of conversion in a manner known way, thus can be for transforming plant expediently.Grow or be provided for plant transformed or plant part in the mode of convention.Make the Agrobacterium of conversion act on plant or plant part subsequently, until reaching enough transfection efficiencies.Can make Agrobacterium act on plant or plant part with multi-form.For example, can use the culture of morphogenetic vegetable cell or tissue.Shift after the T-DNA, bacterium is generally removed by microbiotic, and inducing plant tissue regeneration.This specifically finishes by using suitable plant hormone to grow with initial evoked callus formation and subsequent start-up bud.

[0225.0.0.0] foreign gene is called conversion to the transfer of Plant Genome.For this reason, use described being used to transform and carry out instantaneous or stable conversion from the method for plant tissue or vegetable cell regeneration plant.Favourable method for transformation transforms in planta.For this reason, can for example make Agrobacterium act on plant seed or with Agrobacterium inoculation plant meristematic tissue.According to the present invention, verified make the suspension that transforms Agrobacterium act on complete plant or at least flower primordium be especially easily.Subsequently the continued growth plant (Clough and Bent, Plant J. (1998) 16,735-743) until the seed that obtains the plant of handling.For selecting plant transformed, generally the vegetable material that conversion is obtained places under the selection condition, never to distinguish the conversion plant in the plant transformed.For example, can sow with the seed of aforementioned manner results and at the initial growth after date and suitably select by spraying.Other possibility comprises uses appropriate selection reagent in the seed of growing on the agar plate (in case of necessity after sterilization), so that have only the seed of conversion can grow into plant.Other are favourable (being in particular plant) method for transformation is known and describe to some extent hereinafter for the technician.

[0226.0.0.0] other favourable and suitable methods are by " biological projectile " method (referring to the method as microparticle bombardment), the electroporation of the protoplast transformation of poly-(ethylene glycol) inductive DNA picked-up, use gene big gun, hatch dried embryo, microinjection and agriculture bacillus mediated transgenosis in dna solutions.Described method is described in for example B.Jenes etc., " Transgenic Plants ", the first roll, Engineeringand Utilization, S.D.Kung and R.Wu compile, the Techniques for Gene Transfer of Academic Press (1993) 128-143 and Potrykus Annu.Rev.Plant Physiol.PlantMolec.Biol.42 (1991) 205-225.Nucleic acid to be expressed or construct are preferably cloned and into are fit to transform in the carrier of agrobacterium tumefaciens, for example pBin19 (Bevan etc., Nucl.Acids Res.12 (1984) 8711).To be used for the Agrobacterium that this carrier has transformed subsequently transforming particularly crop plants of plant,, for example the blade smashed to pieces or the blade of chopping will be immersed in the Agrobacterium solution, in suitable medium, cultivate subsequently as tobacco plant by known way.The Plant Transformation that relies on agrobacterium tumefaciens is by for example

With Willmitzer at Nucl.Acid Res. (1988) 16, describe in 9877, or be known in F.F.White in addition, " Transgenic Plants ", the 1st volume, Engineering and Utilization, S.D.Kung and R.Wu compile, Academic Press, 1993, " the Vectors for Gene Transfer in Higher Plants " of 15-38 page or leaf.

[0227.0.0.0] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the sequence or derivatives thereof, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of L-Methionin, L-Threonine and/or L-methionine(Met) biosynthetic pathway of at least one amino acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.0] therefore cultivated spontaneous the mistake and expressed coding and amino acid metabolism (particularly amino acid is synthetic) at least one nucleic acid of related protein or the microorganism of a gene in another embodiment of the present invention.

Other favourable nucleotide sequences that [0229.0.0.0] can express with the combination of sequence that present method is used and/or aforementioned biosynthesis gene are the ATP/ADP translocator sequence described in the WO 01/20009.This ATP/ADP translocator causes raising synth essential amino acid Methionin and/or methionine(Met).In addition, favourable nucleotide sequence that can coexpression is threonine aldolase and/or the lysine decarboxylase of describing in the current state of the art.

[0230.0.0.0] is in another advantageous embodiment of the inventive method, the employed archebiosis of present method suddenlyd change at least one aforementioned gene or an aforementioned nucleic acid, so that compare with mutein not, the activity of respective egg white matter is subjected to metabolite effect less or unaffected fully (particularly not damaging each fine chemicals that produces according to the present invention), or has improved its enzymic activity.Low influence refers to and initial biophase ratio, the regulation and control of enzymic activity are reduced by at least 10%, advantageously at least 20,30 or 40%, particularly advantageously at least 50,60 or 70,80 or 90%, thus obtained as these digital described raisings with the activity of initial biophase than this enzyme.The raising of enzymic activity refers to and initial biophase ratio, improves at least 10%, advantageously at least 20,30,40 or 50%, particularly advantageously at least 60,70,80,90,100,200,300,500 or 1000% enzymic activity.This causes each fine chemicals output of expection of raising.

[0231.0.0.0] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened the degraded methionine(Met) simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.0] in another embodiment of the inventive method, the employed biology of the inventive method be with part reduce or block fully respective egg white matter enzymic activity the mode spontaneous mutation those biologies of at least one aforementioned nucleic acid or aforementioned gene.The reduction of enzymic activity refers to and initial biophase ratio reduction by 10%, advantageously at least 20,30 or 40%, particularly advantageously at least 50,60 or 70%, preferred more enzymic activity at least.

[0233.0.0.0] then must remove the previous mark that transforms or adopt other marks if be intended to some constructs or carrier transformed host cell (particularly vegetable cell) in follow-up conversion.Can be by the method that the technician the is familiar with mark of from host cell (particularly vegetable cell), removing as mentioned below.Particularly, not having the plant of mark (particularly not having antibiotics resistance) is particularly preferred embodiment of the present invention.

[0234.0.0.0] in the methods of the invention, the employed nucleotide sequence of the inventive method advantageously effectively is connected to improve genetic expression with one or more conditioning signals.These are regulated sequence and are intended to different expression gene and protein.Depend on host living beings (as plant or microorganism), this for example can refer to that gene only inducing the back to express and/or cross and express, or be constitutive expression and/or cross expression.These regulate sequences is that for example thereby inductor or repressor combine the sequence that makes its adjusting expression of nucleic acid with it.Except these new adjusting sequences, or as the substituting of these sequences, the natural adjusting of these sequences still may reside in before the practical structures gene, and genetically modified in due course, to close natural adjusting and to improve genetic expression.Yet, be suitable for structurally to simplify as the nucleic acid construct of the present invention of expression cassette (=expression construct=gene construct), that is to say and before the nucleotide sequence or derivatives thereof, do not insert extra conditioning signal, and do not remove natural promoter and adjusting thereof.Replace, so that regulate the mode no longer take place and/or to improve genetic expression natural adjusting series jump.Can also the promotor of these modifications be introduced separately into before the natural gene to improve activity with the form of local sequence (=have the promotor of part nucleotide sequence of the present invention).In addition, gene construct can also advantageously contain one or more known enhancer sequence that effectively are connected with promotor, and they improve the expression of nucleotide sequence.Can also insert extra favourable sequence, for example other regulatory elements or terminator at 3 ' end of dna sequence dna.

The nucleic acid molecule that [0235.0.0.0] code book is invented proteinic nucleic acid molecule and other polypeptide of coding may reside in a nucleic acid construct or the carrier or is present in several.Advantageously, a copy that in nucleic acid construct or carrier, only has nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or its encoding gene.Can be in host living beings several carriers of co expression or nucleic acid construct or carrier.Nucleic acid molecule of the present invention or nucleic acid construct or carrier can insert carrier and be present in the cell with free form.If preferred stable conversion is then used at several and is stablized the carrier that duplicates or insert in the genome from generation to generation.For plant, may take place that plastom is integrated or especially may be that the nuclear gene group is integrated.For inserting more than a gene in host genome, gene to be expressed is present in the gene construct jointly, the carrier of a plurality of genes of for example aforementioned carrying.

The adjusting sequence of [0236.0.0.0] genetic expression rate generally is positioned at nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoding sequence or the segmental upstream of other encoding genes (5 '), and/or downstream (3 ') therebetween.Their concrete control is transcribed and/or the stability of translation and/or transcript.Expression level depends on and being connected of other cell regulation systems, as the protein biosynthesizing and the degeneration system of cell.

[0237.0.0.0] adjusting sequence comprises transcribes and translates adjusting sequence or signal, (physical relationship is transcribed or translation initiation to regulating for example to be positioned at the sequence of upstream (5 '), as promotor or initiator codon) and be positioned at the sequence (physical relationship is transcribed or the stability of translation termination and transcript to regulating, as polyadenylation signal or terminator codon) in downstream (3 ').Except the non-coding region (as intron) of transcribing, regulate sequence and can also be present in the coding region (for example splice site) of transcribing.Adjusting expression of nucleic acids of the present invention and operable promotor are that all can be in the promotor of purpose biology (as microorganism or plant) moderate stimulation genetic transcription in principle in the cell.The suitable promotor that has function in these biologies is well-known.They can be the forms of composing type or inducible promoter.Suitable promotor impels the development-specific in the many cells eukaryote to express and/or tissue specific expression, therefore can advantageously use leaf, root, flower, seed, pore, stem tuber or fruit-specific promoter in plant.

[0238.0.0.0] is as indicated above, and regulating the sequence or the factor can have positive-effect to the introducing expression of gene, thereby improves its expression.Therefore, can transcribe signal (as strong promoter and/or strong enhanser) by force and advantageously strengthen expression by using at transcriptional level.In addition, can also strengthen in translation skill and express, for example by introducing the translational enhancer sequence (as the Ω enhanser, for example improve combining of rrna and transcript) or the stability (for example by replacing 3 ' UTR coding region with known coding 3 ' the UTR zone of giving the transcript high stability of coding) by improving mRNA or stablize transcript by the unstable of elimination transcript, thereby the mRNA molecule translated more continually than wild-type.The element (ARE) and DST (downstream) element that for example are rich in AU in plant make the transcript instability.Residue in mutagenesis verified two conserved domain ATAGAT of research and the GTA district is that the unstable function is necessary.Therefore remove or these elements that suddenly change obviously can cause more stable transcript, higher transcription rate and the protein active of Geng Gao.Transcriptional enhancer can also be " super drive sequences " (Gallie etc., 1987, the Nucl.Acids Research 15:8693-8711) that contains tobacco mosaic virus (TMV) 5 ' untranslated leader and improve protein/RNA ratio.

Enhanser is normally defined and can rely on position and direction and cis-acting elements that stimulated gene is transcribed.The different enhansers that in plant, identified composing type or tissue specificity or stimulated differential stimulus to transcribe.The example that the composing type enhanser is known is enhanser (Odell etc., 1985, Nature 313:810-812) or ocs enhanser (Fromm etc., 1989, PlantCell 1:977:984) from 35S promoter.Other example is for giving the G-frame box motif tetramer (Ishige etc. of high-level constitutive expression in dicotyledonous or monocotyledons, 1999, Plant Journal, 18,443-448) or petE---be rich in A/T sequence (Sandhu etc., 1998 as the quantity enhanser of genetic expression in transgene tobacco and potato plants; Plant Mol Biol.37 (5): 885-96).In addition, miscellaneous cis-acting elements of facilitating specific expressed pattern (for example the inducible expression of biology or abiotic stress is expressed or responded to organ specificity) has been described.Example for provide pathogenic agent or wound-induced express (Rushton, 2002, Plant Cell, 14,749-762) or guard cell specific expressed (Plesch, 2001, Plant Journal 28, element 455-464).

[0239.0.0.0] expresses nucleic acid molecule of the present invention in microorganism favourable adjusting sequence is present in cos, tac, rha, trp, tet, trp-tet, lpp, lac, lpp-lac, the lacI that for example is advantageously used in the gram negative bacterium ^Q-, T7, T5, T3, gal, trc, ara, SP6, λ-P _ROr λ-P _LIn the promotor.Other favourable adjusting sequences for example are present among gram positive bacterium promotor amy, dnaK, xylS and SPO2, yeast or fungal promoters ADC1, MF α, AC, P-60, UASH, MCB, PHO, CYC1, GAPDH, TEF, rp28, the ADH.Particularly advantageous promotor is composing type, tissue or compartment specificity and inducible promoter." promotor " generally is interpreted as the adjusting sequence of the encoding sequence fragment expression of mediation nucleic acid molecule in the nucleic acid molecule in this article.Promotor generally is positioned at encoding sequence fragment upstream.Yet some regulate sequences (for example expression-enhancing elements, as enhanser) also can be arranged in downstream or or even transcriptional domain.

[0240.0.0.0] says in principle, and natural promoter can be used from novel method with its adjusting sequence (as mentioned above those).Can also advantageously additionally or separately use synthetic promoter, particularly when they mediate seed-specific expression described in WO 99/16890.

The employed nucleic acid molecule of [0241.0.0.0] expression present method may need other genes or nucleic acid alone or in combination.Can be by transforming several independently suitable nucleic acid constructs (being expression construct) simultaneously or preferably introducing a plurality of nucleic acid molecule that beneficial gene is expressed of giving by the some expression cassettes of combination on a construct.Can also transform some carriers, progressively in receptor's biology, introduce several expression cassettes at every turn.

[0242.0.0.0] is as indicated above, should advantageously hold the suitable terminator of (after the terminator codon) to stop introducing gene transcription by introducing biosynthesis gene 3 '.The terminator that can be used for this purpose is for example OCS1 terminator, nos3 terminator or 35S terminator.Situation with promotor is identical, and each gene should use different terminator sequences.The terminator that can be used in the microorganism is for example fimA terminator, txn terminator or trp terminator.These terminators can be rho-dependent form or rho independent form.

[0243.0.0.0] can be advantageously uses different plant promoters for example USP, LegB4, DC3 promotor or from ubiquitin promoter or other promotors mentioned in this article and different terminators of Parsley in nucleic acid construct.

[0244.0.0.0] is because the tumor-necrosis factor glycoproteins motif may cause recombination event or reticent or (in plant) T-DNA instability, therefore used nucleic acid molecule stable integration a plurality of generations to the transgenic plant for guaranteeing with the inventive method of other biological synthetic gene combination, each coding region of using in present method should be in expression under himself control of promotor of (preferably exclusive).

[0245.0.0.0] advantageously makes up nucleic acid construct in the following manner: being after the promotor to be preferably the poly joint by suitable cleavage site (being used to insert nucleic acid to be expressed), afterwards is to be positioned at poly joint terminator afterwards in the time of suitably.In the time of suitably, thereby this order can repeated several times make up several genes and so can be incorporated in the transgenic plant to be expressed in a construct.This order advantageously repeats as many as three times.For expressing, insert nucleotide sequence by suitable cleavage site (as the poly joint after the promotor).As indicated above, the terminator that each nucleotide sequence has the promotor of himself and has himself where necessary is favourable.Yet, if can carry out polycistronic transcription in host or the target biology, can also be after a promotor and (suitably time) terminator before insert some nucleotide sequences.In this article, the insertion site in the nucleic acid construct or the order of the nucleic acid molecule that inserts are not conclusive, that is to say that nucleic acid molecule can insert in the box first or last site to expression with having no significant effect.Yet, can also in construct, only use a promotor type.But as described, this may cause undesirable recombination event or reticent effect.

[0246.0.0.0] therefore, in preferred embodiments, nucleic acid construct of the present invention is given nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor and other optional expression of gene in plant, and contains one or more plant regulatory elements.Described nucleic acid construct of the present invention advantageously comprises plant promoter or plant terminators or plant promoter and plant terminators.

[0247.0.0.0] " plant " promotor contains the regulatory element of mediation encoding sequence fragment expression in vegetable cell.Therefore, plant promoter does not need for plant origin, but can derive from virus or microorganism, particularly for example from the virus of attacking vegetable cell.

[0248.0.0.0] plant promoter can also derive from vegetable cell, for example from the plant that has transformed nucleic acid construct or carrier as described herein.This can also be applied to other " plant " conditioning signals, for example " plant " terminator.

The nucleic acid construct that [0249.0.0.0] is suitable for expression of plants preferably contain can controlling gene be expressed in vegetable cell the adjusting sequence, these are regulated sequences and effectively connect so that each sequence can realize its function.Therefore, this nucleic acid construct can also contain transcription terminator.The example of Transcription Termination is a polyadenylation signal.Preferred polyadenylation signal is the sequence that comes from agrobacterium tumefaciens T-DNA, for example be known as the gene 3 (Gielen etc. of the Ti-plasmid pTiACH5 of octopine synthase, 1984, EMBO is J.3:835) or its functional equivalent, but every other the active terminator of function is arranged in plant also is suitable.

The nucleic acid construct that [0250.0.0.0] is suitable for expression of plants preferably also contains the regulatory element that other effectively connect, as translational enhancer, super drive sequences (the Gallie etc. that for example contain the tobacco mosaic virus (TMV) 5 '-untranslated leader of raising protein/RNA ratio, 1987, Nucl.Acids Research15:8693-8711).

[0251.0.0.0] is used for (can consulting Kermode in other preferred sequences that the genetic expression construct effectively connects for the target sequence that gene product target specific cells compartment is required, Crit.Rev.Plant Sci.15, the summary of 4 (1996) 285-423 and the reference of quoting thereof), described compartment is for example outside other compartments or born of the same parents of vacuole, nucleus, all types of plastid (as amyloplast, chloroplast(id), chromoplast), born of the same parents' external space, plastosome, endoplasmic reticulum, elaioleucite, peroxysome, ferment glycolysis body and cell.The sequence that this paper must mention is specially itself known signal peptide or transit peptides encoding sequence.For example the plastid transit peptide-coding sequence makes in the plastid of expression product targeted plants cell.Also known Eukaryotic target sequence and more a spot of procaryotic target sequence can be advantageously effectively be connected obtaining with nucleic acid molecule of the present invention to be expressed outside one of described compartment or born of the same parents.

[0252.0.0.0] for to express in plant, nucleic acid molecule must as indicated abovely effectively be connected with the suitable promotor that gene is expressed in the specific mode of correct time point and cell or tissue, or contains described promotor.Useful promotor is constitutive promoter (Benfey etc., (1989) EMBO J.8:2195-2202), for example derive from the promotor of plant virus, as 35S CAMV (Franck etc., Cell 21 (1980) 285-294), 19SCaMV (consulting US 5352605 and WO 84/02913), 34S FMV (Sanger etc., Plant.Mol.Biol., 14,1990:433-443), Parsley ubiquitin promoter or plant promoter, as U.S. Patent number 4,962, the promotor of the rubisco small subunit described in 028 or plant promoter PRP1 (Ward etc., Plant.Mol.Biol.22 (1993)), SSU, PGEL1, OCS (Leisner (1988) Proc Natl Acad Sci USA85 (5): 2553-2557), lib4, usp, mas (Comai (1990) Plant Mol Biol 15 (3): 373-381), STLS1, ScBV (Schenk (1999) Plant Mol Biol 39 (6): 1221-1230), B33, SAD1 or SAD2 (flax promotor, Jain etc., Crop Science, 39 (6), 1999:1696-1701) or no (Shaw etc. (1984) Nucleic Acids Res.12 (20): 7831-7846).Protein of the present invention stable constitutive expression in plant may be favourable.Yet, if because metabolism operation may cause plant-growth postpones, be useful and later stage before the results is expressed, inducible expression polypeptide of the present invention or the polypeptide that is used for the inventive method are favourable.

[0253.0.0.0] can also the expression (consulting Gatz 1997, Annu.Rev.Plant Physiol.Plant Mol.Biol., the summary of 48:89-108) that promote plant gene by chemical inducible promoter as indicated above.When need be with the mode expressing gene of temporal, chemical inducible promoter be especially suitable.The example of these promotors is the promotor (WO 95/19443) of Induced by Salicylic Acid and dormin inductive promotor (EP 335 528), tsiklomitsin inductive promotor (Gatz etc. (1992) Plant J.2,397-404), hexalin or alcohol induced promotor (WO 93/21334) or other promotors as herein described.

[0254.0.0.0] other suitable promotors are the promotor with biology or abiotic stress conditioned response, PRP1 gene promoter (the Ward etc. of pathogen-inducible for example, Plant.Mol.Biol.22 (1993) 361-366), (US 5 for the thermoinducible hsp80 promotor of tomato, 187,267), the pinII promotor (EP-A-0375091) of the cold inductive αDian Fenmei of potato promotor (WO 96/12814) or wound-induced or other promotors as herein described.

[0255.0.0.0] preferred promotor is specially in amino acid whose tissue of biosynthesizing and organ, make the promotor of genetic expression in the seed cell (as the protoblast of albuminous cell and growth).Suitable promotor is that (US 5 for the rapeseed protein promotor of rape, 608,152), broad bean (Vicia faba) USP promotor (Baeumlein etc., Mol Gen Genet, 1991,225 (3): 459-67), the oleosin promotor (WO 98/45461) of Arabidopis thaliana, the phaseolin promoter (US5 of Kidney bean (Phaseolus vulgaris), 504,200), DcG3 promotor of the arc5 promotor of the Bce4 promotor of rape (WO 91/13980), soybean, Radix Dauci Sativae or legumin B4 promotor (LeB4; Baeumlein etc., 1992, Plant Journal, 2 (2): 233-9) and in monocotyledons (as corn, barley, wheat, rye, rice etc.), cause the promotor of seed-specific expression.Favourable seed specific promoters is sucrose-binding proteins matter promotor (WO 00/26388), phaseolin promoter and rapeseed protein promotor.The suitable promotor that must consider is that the promotor (WO95/15389 and WO 95/23230) of barley lpt2 or lpt1 gene and the promotor described in W099/16890 are (from the hordein gene of barley, the glutenin gene of rice, the paddy rice plain gene of rice, the prolamine gene of rice, the alcohol soluble protein gene of wheat, the glutenin gene of wheat, the zein spirit-soluble gene of corn, the avenaceous glutenin gene, the promotor of kasirin gene of jowar and the secalin gene of naked barley).Other suitable promotors are that (US 5 for Amy32b, Amy6-6 and Aleurain, 677,474), (US 5,530 for Bce4 (rape), 149), glycinin (soybean, EP 571741), phosphoric acid enol pyruvic acid carboxylase (soybean, JP 06/62870), ADR12-2 (soybean, WO 98/08962), isocitrate lyase (rape, US 5,689,040) or α-Dian Fenmei (barley, EP 781 849).Other promotors that can use for expressing gene in plant be described in DE-A19644478 leaf Idiotype promotor or as the light adjusting promotor of pea petE promotor.

[0256.0.0.0] other suitable plant promoters are tenuigenin FBP enzyme promotor or potato ST-LSI promotor (Stockhaus etc., EMBO J.8,1989,2445-245), the tubercle specificity promoter described in soybean Phosphoribosyl tetra-sodium transaminase promotor (Genebank accession number U87999) or the EP-A-0 249 676.

[0257.0.0.0] other particularly suitable promotors are the promotor that causes that plastid is specific expressed.The Arabidopis thaliana clpP promotor of describing among viral rna polymerase promotor of describing among suitable promotor such as WO 95/16783 and the WO 97/06250 and the WO 99/46394.

[0258.0.0.0] is except aforesaid some viruses and bacterium promotor, be used for being preferably Actin muscle or ubiquitin gene plant promoter, as rice Actin muscle 1 promotor in other promotors of tissue as much as possible (particularly also in leaf) strongly expressed heterologous sequence.Other examples of constitutive plant promoters are sugar beet V-ATP enzyme promotor (WO 01/14572).The example of synthetic composing type promoter is a super promotor (Super promoter) (WO 95/14098) and from the promotor (WO 94/12015) of G-frame.In the time of suitably, chemical inducible promoter can also be used in addition, with reference to EP-A 388186, EP-A 335528, WO 97/06268.

[0259.0.0.0] is already mentioned as this paper, and other adjusting sequences that may be suitable for also comprise the sequence of transhipment and/or location targeted expression product when suitable.The sequence that must mention is in particular itself known signal peptide or transit peptides encoding sequence herein.For example, the plastid transit peptide-coding sequence makes in the plastid of expression product targeted plants cell.

[0260.0.0.0] is as indicated above, and preferred recipient plant is specially plant transformed in a suitable manner.Comprise unifacial leaf and dicotyledons.The plant that must specifically mention is the plant (as cereal and grass) of agricultural use, some kind of Triticum (Triticum spp.) for example, Zea mays, barley, oat, rye, rice, cattailmillet (Pennisetum glaucum), dichromatism chinese sorghum, triticale (Triticale), some kind of Agrostis (Agrostis spp.), cilium sandbur (Cenchrusciliaris), orchardgrass (Dactylis glomerata), alta fascue (Festuca arundinacea), some kind of lolium (Lolium spp.), some kind of Medicago (Medicago spp.) and some kind of saccharum (Saccharum spp.), beans and oil crops are leaf mustard for example, colea, soybean, Semen arachidis hypogaeae, upland cotton, garbanzo (Cicer arietinum), Sunflower Receptacle, table tennis beans (Lens culinaris), flax, sinapsis alba (Sinapis alba), white clover (Trifolium repens) and Vicianarbonensis, vegetables and fruit is banana for example, grape, tomato, asparagus, Caulis et Folium Brassicae capitatae, watermelon, Kiwifruit, potato, beet, cassava and witloof are set for example some kind of Coffea (Coffeaspecies), some kind of both citrus (Citrus spp.), eucalyptus belongs to some kind (Eucalyptus spp.), some kind of Picea (Picea spp.), some kind of Pinus (Pinus spp.) and some kind of Populus (Populusspp.), medicinal plant He Shu and flower.

[0261.0.0.0] one embodiment of the invention also relate to the method that produces carrier, and described carrier contains the nucleic acid molecule that this paper characterized that inserts in the oriented carrier, nucleic acid molecule of the present invention or expression cassette of the present invention.For example, as this paper with regard to the description of nucleic acid construct or as hereinafter through conversion or transfection or as shown in embodiment, carrier can be introduced in the cell (as microorganism or vegetable cell).Can instantaneous or stable transfection host or target cell, however preferred stable transfection.Carrier of the present invention is preferably the carrier that is adapted at expressing in the plant polypeptide of the present invention.This method can also comprise and one or more conditioning signal (particularly mediating the signal of expressing in microorganism or plant) is integrated into the step in the carrier.

[0262.0.0.0] therefore the invention still further relates to carrier, and described carrier contains nucleic acid molecule or the nucleic acid molecule of the present invention that conduct that this paper characterizes is fit to the nucleic acid construct part of expression of plants.

The favourable carrier of [0263.0.0.0] the present invention contains the nucleic acid construct that code book is invented proteinic nucleic acid molecule, the employed nucleic acid molecule of the inventive method or suitable expression of plants, described construct contain separately or with other genes (as each fine chemicals biosynthesizing or regulatory gene, such as with gene mentioned above) combination the use nucleic acid molecule.According to the present invention, term " carrier " is meant the nucleic acid molecule that can transport its another nucleic acid that connects.One type carrier is " plasmid ", is meant the circular double stranded DNA ring that can connect extra dna fragmentation.The carrier of another kind of type is a virus vector, and extra nucleic acid fragment can be connected into viral genome.Some carrier can be in the host cell of its introducing self-replicating (bacteria carrier that for example has the bacterium replication origin).Other preferred carriers are incorporated in the host cell gene group after being introduced into host cell wholly or in part, and therefore along with host genome is duplicated together.In addition, some carrier can be controlled and its expression of gene that effectively is connected.In this article, these carriers are known as " expression vector ".As indicated above, they can maybe can be partially or entirely integrated in the host genome by self-replicating.The expression vector that is generally suitable in the DNA recombinant technology often is the plasmid form.Because plasmid is the most frequently used carrier format, so " plasmid " and " carrier " is used interchangeably in this manual.Yet, the invention is intended to comprise the other forms of expression vector of bringing into play similar functions, for example virus vector.This term also comprises other carriers known to the skilled in addition, as phage, virus (as SV40, CMV, TMV), transposon, IS element, phasmid, phagemid, clay and linearity or cyclic DNA.

The recombinant expression vector that [0264.0.0.0] is advantageously used in present method contains nucleic acid molecule of the present invention or nucleic acid construct of the present invention with the form that is adapted at expressing in the host cell nucleic acid molecule of the present invention or nucleic acid molecule described herein.Therefore, this recombinant expression vector contain one or more that select based on the host cell that is used to express and with the conditioning signal for the treatment of that the express nucleic acid sequence effectively is connected.

In recombinant expression vector, nucleic acid molecule of " effectively connect " feeling the pulse with the finger-tip is connected with conditioning signal in the mode that may express this nucleic acid molecule [0265.0.0.0]: they are so that two sequences realize the mode of specified expectation function be connected to each other (for example in in-vitro transcription/translation system or host cell (if carrier is introduced into host cell)).

[0266.0.0.0] term " adjusting sequence " is intended to comprise promotor, enhanser and other expression controlling elementss (for example polyadenylation signal).These regulate sequence at for example Goeddel, " GeneExpression Technology:Methods in Enzymology185 ", Academic Press, San Diego, CA (1990) and Gruber and Crosby, " Methods in PlantMolecular Biology and Biotechnology ", Glick and Thompson compile, the 7th chapter, 89-108, CRC Press:Boca Raton, Florida describes in (comprising the reference of wherein quoting) to some extent.Regulate sequence and comprise that the control nucleotides sequence is listed in the sequence of constitutive expression in the broad variety host cell, and the sequence that only in particular host cell and under the specified conditions, instructs nucleotide sequence to express.The design that skilled person in the art will appreciate that expression vector is based on these factors, as the selection of host cell to be transformed, the expression of polypeptides level that needs etc.Preferred adjusting sequence selection has above been described, for example promotor, terminator, enhanser etc.Term adjusting sequence should be thought of as and be included in the term conditioning signal.Some favourable adjusting sequence, particularly promotor and terminators have above been described.The adjusting sequence that is described as helping being fit to the nucleic acid construct of expressing generally also is applicable to carrier.

[0267.0.0.0] can design employed recombinant expression vector especially, to express the employed nucleic acid molecule of present method in protokaryon and/or eukaryotic cell.Because the intermediate steps of vector construction is often considered for simplicity and is carried out in microorganism, so this is favourable.For example, gene of the present invention and other genes can use carrier and (see Romanos according to method for transformation described in the WO 98/01572 bacterial cell, insect cell (use rhabdovirus expression vector), yeast and other fungal cells, 1992, Yeast 8:423-488; Van den Hondel, (1991), " More GeneManipulations in Fungi ", J.W.Bennet ﹠amp; L.L.Lasure compiles 396-428 page or leaf, Academic Press:San Diego; With van den Hondel, C.A.M.J.J. (1991), " Applied Molecular Genetics of Fungi, Peberdy ", volumes such as J.F., 1-28 page or leaf, Cambridge University Press:Cambridge), algae (Falciatore etc., 1999, Marine Biotechnology 1 (3): express 239-251), preferably (consult Schmidt at the metaphyte cell, R. and Willmitzer, L., (1988) Plant Cell Rep.583-586; " PlantMolecular Biology and Biotechnology ", C Press, Boca Raton, Florida, 6/7 chapter, 71-119 page or leaf (1993); F.F.White, " Transgenic Plants ", the 1st volume, " Engineering and Utilization ", Kung and R.Wu compile, Academic Press (1993), 128-43; Potrykus, Annu.Rev.Plant Physiol.Plant Molec.Biol.42 (1991), 205-225 and the reference of quoting thereof) the middle expression.Proper host cell is at Goeddel, and " Gene Expression Technology:Methods in Enzymology 185 ", AcademicPress:San Diego, CA has further discussion in (1990).Alternatively, the sequence of recombinant expression vector can in-vitro transcription and translation, for example uses the T7 promotor to regulate sequence and T7 polysaccharase.

[0268.0.0.0] can use and contain control fusion rotein or the composing type of non-expressing fusion protein or carrier marking protein in prokaryotic organism of inducible promoter.Typical fusion expression vector is pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. pMAL (New England Biolabs (1988) Gene67:31-40),, Beverly, MA) and therein pRIT5 (the Pharmacia that merges of target recombinant protein and glutathione S-transferase (GST), maltose-E-conjugated protein and A albumen, Piscataway, NJ).The example of the non-fusion coli expression carrier of suitable induction type is pTrc (Amann etc., Gene 69:301-315) and pET 11d carrier [Studier etc. (1988), " Gene Expression Technology:Methods in Enzymology 185 ", Academic Press, San Diego, California (1990) 60-89].The expression of target gene of pTrc carrier is based on by host RNA polysaccharase transcribing heterozygosis trp-lac promoter, fusion.Transcribe based on the T7-gn10-lac promoter, fusion that the viral rna polymerase (T7gn1) by coexpression mediates from pET 11d vector expression target gene.This varial polymerases is provided from the λ prophage of settling down that carries T7 gn1 gene under the control of lacUV5 promoter transcription by host strain BL21 (DE3) or HMS174 (DE3).

[0269.0.0.0] is fit to procaryotic other carriers is that the technician is known, and these carriers are pLG338, pACYC184, pBR series (as pBR322), pUC series (as pUC18 or pUC19), M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, the pIN-III in the intestinal bacteria for example ¹¹³-B1, λ gt11 or pBdCl; PIJ101 in the streptomycete, pIJ364, pIJ702 or pIJ361; PUB110 in the genus bacillus, pC194 or pBD214; PSA77 or pAJ667 in the rod bacillus.

[0270.0.0.0] in another embodiment, expression vector is a Yeast expression carrier.The example that is used for the expression vector of yeast saccharomyces cerevisiae comprises pYeDesaturasec1 (Baldari, Deng, (1987) Embo is J.6:229-234), pMFa (Kurjan and Herskowitz, (1982) pJRY88 (Schultz etc. Cell 30:933-943),, Gene 54:113-123) and pYES2 (InvitrogenCorporation (1987), San Diego, CA).The carrier and the carrier construction method that are applicable to other fungies (as filamentous fungus) are included in van den Hondel C.A.M.J.J.[(1991), J.F.Peberdy compiles, 1-28 page or leaf, Cambridge University Press:Cambridge; Perhaps at " More GeneManipulations in Fungi ", J.W.Bennet and L.L.Lasure compile, 396-428 page or leaf: those that describe in detail among the Academic Press:San Diego.The example of the yeast vector that other are fit to is 2 μ M, pAG-1, Yep6, Yep13 or pEMBLYe23.

Other carriers that [0271.0.0.0] may mention as an example are pALS1, the pIL2 in the fungi or pLGV23, the pGHlac in pBB116 or the plant for example ⁺, pBIN19, pAK2004 or pDH51.

[0272.0.0.0] alternatively can use rhabdovirus expression vector at the expressed in insect cells nucleotide sequence.The baculovirus vector that is used in the middle marking protein of insect cell (for example Sf9 cell) of cultivation comprises pAc series (Smith etc., (1983) Mol.Cell Biol.3:2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0273.0.0.0] aforementioned bearer is the simplified summary of possibility suitable carriers just.Other plasmids are well known to those skilled in the art and describe to some extent in for example " Cloning Vectors " volumes such as (, Elsevier, Amsterdam-New York-Oxford, 1985 ISBN 0 444 904018) Pouwels P.H..Be fit to protokaryon and eukaryotic other expression systems and can consult Sambrook, J., Fritsch, E.F. and Maniatis, T., " Molecular Cloning:A Laboratory Manual ", second edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 the 16th and 17 chapters.

[0274.0.0.0] therefore, one embodiment of the invention relate to nucleic acid molecule wherein of the present invention and regulate the carrier that sequence effectively is connected, described adjusting sequence permission is expressed in protokaryon or eucaryon host or in protokaryon and the eucaryon host.

[0275.0.0.0] therefore, one embodiment of the invention relate to the host cell of stable or instantaneous conversion carrier of the present invention or nucleic acid molecule of the present invention or nucleic acid construct of the present invention.

[0276.0.0.0] depends on host living beings, cultivates or the employed biology of the inventive method of growing in the mode that the technician is familiar with.Generally under the temperature between 0 ℃ and 100 ℃ (preferred 10 ℃ and 60 ℃), when flowing oxygen supply contain carbon source (normal form) for sugar, nitrogenous source (often is the form of organic nitrogen source, as yeast extract), or culturing micro-organisms in the liquid nutrient medium of salt (as ammonium sulfate), trace element (as molysite, manganese salt, magnesium salts) and the VITAMIN suitably the time.For microorganism is the situation of anerobe, the obstructed oxygen of substratum.The pH value of liquid nutrient media can keep constant (that is to say in culturing process and regulate) otherwise or.The formula of can criticizing is cultivated, half batch of formula cultivated or the cultured continuously biology.Nutrition can fermentation begin provide semi-continuous or lasting and replenish.

[0277.0.0.0] can separate the amino acid that produces by the method that the technician is familiar with from biology.For example by extraction, salt precipitation and/or ion-exchange chromatography etc.Can destroy biology earlier for this reason.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.Known cultivation and isolation technique are found in Chmiel[Bioproze β technik 1, Einf ü hrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)], (IndustrialMicrobiology and Biotechnology such as Demain, second edition, ASM Press, Washington, D.C., 1999, ISBN 1-55581-128-0) textbook or Storhas (Bioreaktoren undperiphere Einrichtungen) (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)) textbook.

[0278.0.0.0] the present invention relates to the polypeptide by nucleic acid molecule encoding of the present invention in one embodiment, and described polypeptide preferably gives biology after improving its expression or activity or each fine chemicals content of cell improves.

[0279.0.0.0] the invention still further relates to the method that produces polypeptide of the present invention, and described polypeptide is expressed in host cell of the present invention (preferred microorganism or transgenic plant cells).

[0280.0.0.0] in one embodiment, in producing the method for polypeptide employed nucleic acid molecule from microorganism, preferably from protokaryon or protozoan cell, and with eukaryote as host cell.For example, in one embodiment, use nucleic acid molecule in vegetable cell or plant, to produce this polypeptide from protokaryon or fungi or algae or another microorganism (but not being) from plant.

[0281.0.0.0] technician understands; although expressed protein and DNA have same encoding sequence in different biologies; but there are differences in many aspects and on the characteristic (for example DNA regulation and control (modulation) and the marking); for example methylate or posttranslational modification, as glycosylation, phosphorylation, acetylize, myristoylation, ADP ribosylation, farnesylation, carboxylation, sulfation, ubiquitinization etc.Preferably, the cell expressing of respective egg white matter is controlled on the controlling mechanism of the activity of control endogenous protein or another eukaryotic protein and expression and correspondingly there are differences.A main difference of protokaryon or eukaryote marking protein is glycosylated quantity and pattern.For example in intestinal bacteria, there is not glycosylated protein.Expressed protein has high mannose content in the yeast in glycosylated protein, and glycosylation pattern is complicated in plant.

[0282.0.0.0] preferably produces polypeptide of the present invention by recombinant DNA technology.For example, the cloned nucleic acid molecule of coded protein is advanced carrier (as indicated above), carrier is introduced in the host cell (as indicated above) and express described polypeptide in host cell.Can use standard protein purification technique from cell, to separate described polypeptide by suitable purification scheme then.Except recombinant expressed, can use standard peptide synthetic technology chemosynthesis polypeptide of the present invention or peptide.

[0283.0.0.0] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-as Table II the 3rd row, 1-5 is capable and/or 334-338 capable shown in proteinic antibody, for example anti-as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the antibody of polypeptide, polypeptide (polypeptide for example of the present invention or its fragment) generation that it can comprise above-mentioned sequence or be made up of above-mentioned sequence by the standard technique utilization.Preferably with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptid specificity bonded monoclonal antibody.

[0284.0.0.0] the present invention relates to have the polypeptide of the coded aminoacid sequence of the nucleic acid molecule that obtains by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or by the inventive method in one embodiment.Described polypeptide is preferably given aforementioned activity, and particularly, polypeptide is given each fine chemicals increase in cell or biological or its part after (for example by improving this polypeptide expression or specific activity) improves cytoactive.

[0285.0.0.0] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence or by as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.0.0] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in the capable and/or 334-338 of 1-5 is capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in the capable and/or 334-338 of 1-5 is capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, 1-5 is capable and/or 334-338 capable shown in the polypeptide of (different rows) consensus sequence.

[0287.0.0.0] in one embodiment, be no more than 15%, preferred 10% even more preferably 5%, 4%, 3% or 2%, most preferably 1% or 0% alphabetical specified amino acid position by another aminoacid replacement or, in another embodiment, lacked and/or replaced.In another embodiment, by (X) _n20%, preferred 15 or 10% of its length of extension of the non-conserved amino acid that [wherein n represents the number of X] is represented, even be more preferably 5%, 4%, 3%, 2% or 1% difference only most preferably.

[0288.0.0.0] in one embodiment, inserted in the consensus sequence 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4 even more preferably 3 even more preferably 2 even more preferably 1, most preferably 0 amino acid or, in another embodiment, amino acid is lacked and/or is replaced.

It is right that the consensus sequence that shows in [0289.0.0.0] literary composition comes from as the multiple ratio of the sequence of listing in the Table II.The consensus sequence in ad hoc structure territory is right from the multiple ratio of all sequences.The letter single letter amino acid code of representative also shows that this amino acid is guarded in the protein of all comparisons.Letter X representative nonconservative amino acid in all sequences.In an example, only may there be little selected amino acid subclass in some cases in a certain position, in bracket, have provided these amino acid.The number of the X that provides is represented the distance between the conservative amino acid residues, and for example YX (21-23) the F meaning is that tyrosine conservative in the sequence of full-fledged research and phenylalanine residue are separated by minimum 21 and maximum 23 amino-acid residues each other.

[0290.0.0.0] uses Vector NTI Suite 8.0, (InforMax ^TM, Invitrogen ^TMLifescience software, U.S.Main Office, 7305Executive Way, Frederick, MD21704, U.S.) assembly AlignX software (on September 25th, 2002) and use following setting to compare: for the pairing comparison: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Right for multiple ratio: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Breach separates the point penalty scope: 8; Residue is replaced matrix: blosum62; Hydrophilic residue: G P S N D Q E K R; Conversion weight: 0.5; Total calculation options: total residue mark: 0.9.Also selected to set in advance in order to compare conserved amino acid.

[0291.0.0.0] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise and contain plant or microorganism specificity consensus sequence or by its polypeptide of forming.In one embodiment, described polypeptide of the present invention by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in the capable and/or 334-338 of 1-5 is capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in the capable and/or 334-338 of 1-5 is capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence form.

[0292.0.0.0] in one embodiment, polypeptide of the present invention contains previous one of the sequence known to the public that is not.In one embodiment, polypeptide of the present invention derives from non-plant cell (particularly from microorganism), and expresses in vegetable cell.In one embodiment, the present invention relates to by polypeptide nucleic acid molecule of the present invention or nucleic acid molecule encoding that the inventive method is used, that its activity is not described as yet.

[0293.0.0.0] the present invention relates to give the polypeptide that each fine chemicals increases in biological or its part in one embodiment, and described polypeptide is by nucleic acid molecule of the present invention or nucleic acid molecule encoding that the inventive method is used.

In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in other sequence of sequence phase region.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence form.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help as Table I A or IB the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the sequence of nucleic acid molecule encoding form.

[0294.0.0.0] in one embodiment, the present invention relates to have as Table II the 3rd row, the polypeptide of activity of proteins shown in the capable and/or 334-338 of 1-5 is capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in the capable and/or 334-338 of 1-5 is capable.

The term " protein " and " polypeptide " that use among [0295.0.0.0] the application are interchangeable." polypeptide " relates to aminoacid polymers (aminoacid sequence), do not relate to the length-specific of molecule.Therefore peptide and oligopeptides are contained in the definition of polypeptide.This term also relates to or comprises the posttranslational modification of polypeptide, for example glycosylation, acetylize, phosphorylation etc.The polypeptide that contains one or more amino acid analogues (for example comprise alpha-non-natural amino acid etc.) that defines that included for example natural existence or non-natural exist, has replacement key (Substituted linkage) and known in the art other are modified the polypeptide of (comprising modification natural or that non-natural takes place).

[0296.0.0.0] polypeptide is preferably isolating." isolating " or " purifying " protein or nucleic acid molecule or its biologically-active moiety do not contain cellular material (when producing by recombinant DNA technology) or precursor or other chemical substances (during chemosynthesis) substantially.

The statement that [0297.0.0.0] " do not contain cellular material " substantially comprises the wherein preparation of protein isolating polypeptide of the present invention from the cellular component of natural or its cell of reorganization generation.In one embodiment, the statement that " does not contain cellular material substantially " comprises and contains the preparation that is lower than about 30% (dry weight) " contaminating protein matter ", " the contaminating protein matter " more preferably less than about 20%, also more preferably less than about 10% " contaminating protein matter ", and most preferably be lower than the preparation of about 5% " contaminating protein matter "." contaminating protein matter " relates to the polypeptide that is not polypeptide of the present invention to term.When reorganization produces polypeptide of the present invention or its biologically-active moiety, also preferably do not contain substratum substantially, promptly substratum less than the protein formulation cumulative volume 20%, be more preferably less than 10%, and most preferably less than 5%.The statement that " does not contain precursor or other chemical substantially " comprises polypeptide wherein of the present invention isolating preparation from the precursor that participates in protein synthesis or other chemical.The statement that " does not contain precursor or other chemical substantially " comprises and contains the precursor that is lower than 30% (dry weight) or the chemical of non-polypeptide of the present invention, preferably be lower than 20% precursor or non-chemiluminescent polypeptide product of the present invention, also more preferably less than 10% precursor or non-chemiluminescent polypeptide product of the present invention, and most preferably be lower than 5% the precursor or the preparation of non-chemiluminescent polypeptide product of the present invention.In preferred embodiments, isolating protein or its biologically-active moiety do not derive from the contaminating protein matter of the same biology that produces polypeptide of the present invention.Generally produce this proteinoid by recombinant technology.

The chemical of [0297.1.0.0] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 1-5 is capable and/or 334-338 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.

[0298.0.0.0] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the identical aminoacid sequence of sequence.

[0299.0.0.0] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in one of nucleotide sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.0.0] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,1-5 is capable and/or 334-338 capable shown in sequence different.Therefore, this polypeptide contain with as Table II A or IIB the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.0] is the comparing amino acid sequence, can use same algorithm or nucleotide sequence as indicated above.Use Needleman and Wunsch or Smith and Waterman algorithm to obtain high quality results.Therefore be preferably based on the program of described algorithm.Can service routine PileUp (J.Mol.Evolution., 25,351-360,1987, Higgins etc., CABIOS, 5 1989:151-153) or preferred service routine Gap and BestFit comparative sequences advantageously, they are respectively based on the algorithm (J.Mol.Biol.48 of Needleman and Wunsch; 443-453 (1970)) and Smith and Waterman (Adv.Appl.Math.2; 482-489 (1981)) algorithm.Two programs all be the GCG software package (Genetics Computer Group, 575Science Drive, Madison, Wisconsin, USA 53711 (1991); Altschul etc. (1997) Nucleic Acids Res.25:3389 and following or the like) a part.Therefore preferably on the complete sequence scope, use the Gap program to finish calculating to determine sequence homology per-cent.Nucleotide sequence relatively used following standard adjustment: breach weight: 8; Length weight: 2; Average coupling: 2.912; Average mispairing :-2.003.

The biologically-active moiety of [0302.0.0.0] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence shown in the capable and/or 334-338 of 1-5 is capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.0] usually, biological (or immunity) active part (be peptide, for example length be 5,10,15,20,30,35,36,37,38,39,40,50,100 or the peptide of amino acids more) contain the activity with at least a polypeptide of the present invention or polypeptide that the inventive method is used or the structural domain or the motif of epi-position.In addition, can wherein lack other regional other biological active parts of polypeptide by the recombinant technology preparation, and assess one or more activity as herein described.

[0304.0.0.0] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as Table II the 3rd row, 1-5 is capable and/or 334-338 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.0] causes that polypeptide of the present invention or polypeptide that the inventive method is used improve described active any mutagenesis strategy and all be meant nonrestrictively, and the variation of these strategies is that those skilled in the art are conspicuous.Use these the strategy and in conjunction with mechanism disclosed herein, the polypeptide that can utilize nucleic acid molecule of the present invention and polypeptide or be used for the inventive method produces plant or its part, described plant or its part are expressed the nucleic acid molecule of the present invention or the peptide molecule of one or more wild-type proteins of coding or one or more muteins, with yield, output and/or the generation efficient that improves required compound.

[0306.0.0.0] this required compound can be any natural product of plant, comprises in the intermediate product of the end product of biosynthetic pathway and naturally occurring pathways metabolism and the not natural metabolism that is present in described cell but the molecule that produced by cell of the present invention.Preferably, compound is the composition of each fine chemicals of comprising each fine chemicals or recovery (particularly free or with the fine chemicals of protein bound form).

[0306.1.0.0] preferably, compound is the composition of the methionine(Met) that comprises methionine(Met) or recovery (particularly free or with the fine chemicals of protein bound form).

[0307.0.0.0] the present invention also provide chimeric or fusion rotein.

[0308.0.0.0] is as used herein, " chimeric protein " or " fusion rotein " comprises the polypeptide that effectively is connected with following polypeptide, described polypeptide is not given above-mentioned activity after its activity is enhanced, particularly do not give each fine chemicals content raising in cell or biological or its part.

[0309.0.0.0] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, be meant the polypeptide that has corresponding polypeptide of the present invention or be used for the amino acid sequence of polypeptide of the inventive method shown in the capable and/or 334-338 of 1-5 is capable, be not listed as and be shown in Table II the 5th or 7, the polypeptide that " non-polypeptide of the present invention " or " other polypeptide " during the capable and/or 334-338 of 1-5 is capable is meant the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with as Table II the 5th or 7 be listed as, polypeptide shown in the capable and/or 334-338 of 1-5 is capable is homology not basically, for example do not give described in the literary composition active or as Table II the 3rd be listed as, note shown in the capable and/or 334-338 of 1-5 is capable or known and from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, 1-5 is capable and/or " the non-of the present invention polypeptide " or " other polypeptide " of 334-338 in capable do not given each fine chemicals in organism or its part increase.

In fusion rotein, term " effectively connection " is intended to refer to that polypeptide of the present invention or polypeptide that the inventive method is used merge each other with " other polypeptide " or its part, makes two sequences all bring into play the expectation function of the sequence of using [0310.0.0.0]." other polypeptide " can be merged N end or C end to polypeptide of the present invention or polypeptide that the inventive method is used.For example, in one embodiment, fusion rotein is the GST-LMRP fusion rotein, and the sequence of polypeptide wherein of the present invention or polypeptide that the inventive method is used merges the C end to the GST sequence.Such fusion rotein can be beneficial to the purifying of recombinant polypeptide useful in recombinant polypeptide of the present invention or the inventive method.

[0311.0.0.0] in another embodiment, fusion rotein contains in the polypeptide of the present invention of allos signal sequence or the inventive method by N end and is used polypeptide.In some host cell (for example mammalian host cell), can improve the polypeptide expression of using and/or secretion in polypeptide of the present invention or the inventive method by using the allos signal sequence.State as mentioned, the target sequence is necessaryly (can consult Kermode to gene product target specific cell compartment, Crit.Rev.Plant Sci.15, the summary of 4 (1996) 285-423 and the reference of quoting thereof), described compartment is for example outside other compartments or born of the same parents of vacuole, nucleus, all types of plastid (as amyloplast, chloroplast(id), chromoplast), born of the same parents' external space, plastosome, endoplasmic reticulum, elaioleucite, peroxysome, glycolysis enzyme body and cell.The sequence that this paper must mention is in particular itself known signal peptide or transit peptides encoding sequence.For example the plastid transit peptide-coding sequence makes in the plastid of expression product targeted plants cell.Also known Eukaryotic target sequence and more a spot of procaryotic target sequence, and they can be advantageously effectively be connected obtaining with nucleic acid molecule of the present invention and express outside one of described compartment or born of the same parents.

[0312.0.0.0] preferably produces chimeric or fusion rotein of the present invention by the standard recombinant dna technology.For example, encode the dna fragmentation of different peptide sequences according to linking together in the routine techniques frame, for example flat end or sticking end are used to connect by using, digestion with restriction enzyme with produce suitable end, suitably mend flat sticky end, alkaline phosphatase treatment connects to prevent unexpected connection and enzyme.Can comprise that the automatization dna synthesizer synthesizes fusion gene by routine techniques.Alternatively, can use anchor primer to carry out the pcr amplification of gene fragment, this produces the complementary overhang between two continuous gene fragments, with after annealing and increase again and (for example see " Current Protocols in Molecular Biology " to produce chimeric gene sequence, volumes such as Ausubel, John Wiley ﹠amp; Sons:1992).In addition, the many expression vectors that merge part (for example gst polypeptide) of having encoded are that commerce can get.Thereby nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor can be cloned into such expression vector will merge in part and the encoded protein matter frame and be connected.

[0313.0.0.0] in addition, the segmental mold that can use suitable computer program to carry out the proteinic structural motif of the present invention fit computer redesign (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput.Appl.Biosci.11 (1995), 675-679).Can use the computer model of protein folding to carry out conformation and energy spectrometer (Monge, J.Mol.Biol.247 (1995), the 995-1012 of detailed peptide and protein model; Renouf, Adv.Exp.Med.Biol.376 (1995), 37-45).Can use suitable procedure by the computer aided search of complementary peptide sequences identify in polypeptide of the present invention or the inventive method the interaction sites (Fassina between the polypeptide that uses and its substrate or binding factor or other interacting proteins, Immunomethods (1994), 114-120).Other are used to design the suitable computer system description to some extent in the prior art of protein and peptide, for example at Berry, and Biochem.Soc.Trans.22 (1994), 1033-1036; Wodak, Ann.N.Y.Acad.Sci.501 (1987), 1-13; Pabo, Biochemistry 25 (1986), among the 5987-5991.The result who obtains from the analysis of aforementioned calculation machine can be used for for example preparing protein of the present invention or its segmental peptide mimics.The plan peptide analogs of this protein natural acid sequence can simulate very effectively parent's protein (Benkirane, J.Biol.Chem.271 (1996), 33218-33224).For example, the achirality Q amino-acid residue that obtains easily is integrated into causes amido linkage to be replaced in protein of the present invention or its fragment by the polymethylene unit of aliphatic chain, thereby the convenient strategy that makes up peptide mimics is provided, and (Banerjee, Biopolymers 39 (1996), 769-777).

The description to some extent in the prior art of the potent simulating peptide analogue of oligopeptide hormone in [0314.0.0.0] other system (Zhang, Biochem.Biophys.Res.Commun.224 (1996), 327-331).Also can identify the proteinic appropriate peptide stand-in of the present invention by the compound (for example their combination and immune property) that produces via synthetic peptide mimics combinatorial library of successive alkylation of amide and detection.Production method of peptide mimics combinatorial library and uses thereof is described in the prior art to some extent, for example at Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg.Med.Chem.4 (1996) describes among the 709-715 to some extent.

[0315.0.0.0] in addition, proteinic three-dimensional of the present invention and/or crystalline structure can be used for designing the bioactive peptide mimics inhibition of protein of the present invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg.Med.Chem.4 (1996), 1545-1558).

[0316.0.0.0] in addition, proteinic three-dimensional of the present invention and/or crystalline structure and polypeptide of the present invention or the used polypeptide of the inventive method and the evaluation of its substrate or binding factor interaction sites be can be used for identifying or design combine or change the active mutant that is conditioned.For example, can simulate the active centre and the amino-acid residue of regulating the participation catalyzed reaction of polypeptide of the present invention, to improve or to reduce the substrate combination to activate or the improvement polypeptide.Amino acid whose evaluation to active centre and participation catalyzed reaction promotes screening to have the active mutant of raising.

[0317.0.0.0] sequence shown in Table I-IV the 5th row in the text also describes in gene/ORF locus title in Table I, II, III or IV the 3rd row.

[0318.0.0.0] in an especially preferred embodiment, polypeptide of the present invention do not have in addition with shown in the known gene of listing/ORF locus title or as table in the 3rd row those coded proteinic sequences of described sequence.

[0319.0.0.0] one embodiment of the invention also relate to the antibody of specific combination polypeptide of the present invention or part (i.e. this protein specific fragment or epi-position).

[0320.0.0.0] can use antibody of the present invention to identify and be separated in polypeptide of the present invention and the encoding gene for preparing in the plant described in the literary composition in arbitrarily biological (preferred plant).But these antibody monoclonal antibodies, polyclonal antibody or synthetic antibody and antibody fragment are as Fab, Fv or scFv fragment etc.Monoclonal antibody can be by being described at first

And Milstein, Nature 256 (1975), 495, and Galfr6, Meth.Enzymol.73 (1981), the technology preparation in 3, it comprise murine myeloma cell be derived from the fusion of immune mammiferous splenocyte.

[0321.0.0.0] in addition, the antibody of above-mentioned peptide or its fragment can be by using for example Harlow and Lane " Antibodies, A Laboratory Manual ", CSH Press, Cold SpringHarbor, the method described in 1988 obtains.Can use this antibody to carry out the synthetic of the proteinic immunoprecipitation of the present invention and immunolocalization and this protein of monitoring (for example in the reorganization biology), and the compound of evaluation and protein interaction of the present invention.For example can use the surperficial plasmon resonance of using in the BlAcore system to improve the phage antibody efficiency of selection, from the single library of phage antibody, produce the antibody (Schier that the avidity height increases in conjunction with protein epitope of the present invention, HumanAntibodies Hybridomas 7 (1996), 97-105; Malmborg, J.Immunol.Methods 183 (1995), 7-13).In many cases, antibody is equivalent to combining of other parts/anti-part with antigenic fixation phenomenon.

[0322.0.0.0] the present invention relates to comprise the antisense nucleic acid molecule of the complementary sequence of nucleic acid molecule of the present invention in one embodiment.

[0323.0.0.0] modifies expression level and/or active method is that those skilled in the art are known, and for example comprise crossing and express, suppress altogether, use ribozyme, justice and antisense strategy or other gene silencing approach that for example RNA disturbs (RNAi) or promoter methylation." positive-sense strand " refers in the double chain DNA molecule and its mRNA transcript homologous chain." antisense strand " comprises and " positive-sense strand " complementary reversing sequence.In addition, can modify expression level and/or activity by adjusting or coding region introducing sudden change at nucleic acid of the present invention.In addition, can express the specific combination desired polypeptides and also therefore block its active antibody.The protein bound factor can be for example fit (Famulok M and Mayer G (1999) Curr Top Microbiol Immunol 243:123-36) or antibody or antibody fragment or single-chain antibody.Obtain that the mode of these factors is existing to be described and be that the technician is known.For example, utilize tenuigenin scFv antibody in genetically modified tobacco plant, to regulate phytochrome A activity of proteins (Owen M etc., (1992) Biotechnology (NY), 10 (7): 790-794; Franken E etc., (1997) Curr Opin Biotechnol 8 (4): 411-416; Whitelam, (1996) TrendPlant Sci 1:286-272).

[0324.0.0.0] " antisense " nucleic acid molecule comprises " justice " nucleic acid molecule complementation with coded protein, for example with double-stranded cDNA molecule encoding chain complementary or with coding mRNA sequence complementary nucleotide sequence.Therefore, antisense nucleic acid molecule can combine with just nucleic acid molecule by hydrogen bond.Antisense nucleic acid molecule can with the whole coding strand of nucleic acid molecule of giving the expression of polypeptides that uses in polypeptide of the present invention or the inventive method (as nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding strand) complementation, or only with its part complementation.Therefore, antisense nucleic acid molecule can be the antisense strand of the nucleotide sequence coded chain of nucleic acid molecule of the present invention " coding region ".Term " coding region " refers to contain the nucleotide sequence district of the codon of translating into amino-acid residue.In addition, antisense nucleic acid molecule by in code book invention polypeptide or the inventive method the antisense strand of " non-coding region " of nucleotide sequence coded chain of use polypeptide.Term " non-coding region " refers to that the both sides, coding region do not translate into amino acid whose 5 ' and 3 ' sequence, promptly be also referred to as 5 ' and 3 ' non-translational region (5 '-UTR or 3 '-UTR).

The coding strand sequence of [0325.0.0.0] given code book invention polypeptide can be according to Watson and Crick basepairing rule design antisense nucleic acid molecule of the present invention.

[0326.0.0.0] antisense nucleic acid molecule can with the complete coding region complementation of the mRNA of code book invention nucleic acid molecule or nucleic acid molecule that the inventive method is used, but only also can be a part of complementary oligonucleotide with this mRNA coding region or non-coding region.For example, antisense oligonucleotide can with described mRNA translation initiation site around regional complementarity.The length of antisense oligonucleotide can be for example about 5,10,15,20,25,30,35,40,45,50,100 or 200 Nucleotide.Can use methods known in the art, utilize chemosynthesis and enzyme ligation to make up antisense nucleic acid molecule of the present invention or be used for the antisense nucleic acid molecule of the inventive method.For example, can use the synthetic antisense nucleic acid molecule (for example antisense oligonucleotide) of Nucleotide chemistry of naturally occurring Nucleotide or various modifications, the Nucleotide that designs described modification is for the biologically stable that improves molecule or improves antisense and the physical stability of the duplex that forms between phosphorothioate odn is arranged, the Nucleotide that for example can use thiophosphoric acid derivative and acridine to replace.The Nucleotide example that can be used to produce the modification of antisense nucleic acid comprises 5 FU 5 fluorouracil, 5-bromouracil, the 5-chlorouracil, 5-iodouracil, xanthoglobulin, xanthine, the 4-acetylcytosine, 5-(carboxyl hydroxymethyl) uridylic, 5-carboxyl methylamino methyl-2-sulphur uridine, 5-carboxyl methylamino 6-Methyl Uracil, two hydrogen uridylics, β-D-galactosylqueosine, Trophicardyl, the N6-isopentennyladenine, the 1-methyl guanine, the 1-methyl inosine, 2, the 2-dimethylguanine, the 2-methyladenine, the 2-methyl guanine, the 3-methylcystein, 5-methylcytosine, the N6-VITAMIN B4, the 7-methyl guanine, 5-methylamino 6-Methyl Uracil, 5-methoxyl group amino methyl-2-thiouracil, β-D-mannosylqueosine, 5 '-methoxyl group carboxyl 6-Methyl Uracil, the 5-methoxyuracil, 2-methylthio group-N6-isopentennyladenine, uridylic-the 5-ethoxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-sulfo-cytosine(Cyt), 5-methyl-2-deracil, the 2-deracil, the 4-deracil, methyl uracil, uridylic-5-oxy acetic acid methyl ester, uridylic-the 5-fluoroacetic acid (v), 5-methyl-2-deracil, 3-(3-amino-3-N-2-carboxyl propyl group) uridylic, (acp3) w and 2,6-diaminopurine.In addition, can use the expression vector that the nucleic acid subclone is arrived wherein with antisense orientation (promptly will be the antisense orientation of purpose target nucleic acid, specific descriptions are hereinafter arranged) to come biological generation antisense nucleic acid from the RNA that inserts the transcribed nucleic acid generation.

[0327.0.0.0] antisense nucleic acid molecule of the present invention is given cell usually or original position produces, improve active polypeptide of the present invention or be used for the mRNA of polypeptide and/or the genomic dna hybridization of the inventive method or combine thereby they have above-mentioned each fine chemicals with intracellular coding, thereby for example suppress this protein expression by suppressing to transcribe and/or translate.

[0328.0.0.0] can form stable duplex by conventional Nucleotide is complementary, perhaps for example for DNA duplex bonded antisense nucleic acid molecule, hybridize by the special interaction in double-helical major groove.Can also use carrier described herein that antisense nucleic acid molecule is sent and enter cell.For making antisense molecule reach enough intracellular concentrations, preferably antisense nucleic acid molecule is placed the carrier under the control of strong protokaryon, virus or eucaryon (comprising plant) promotor.

[0329.0.0.0] in another embodiment, antisense nucleic acid molecule of the present invention or the antisense nucleic acid molecule that is used for the inventive method are α-end group isomery nucleic acid molecule.α-end group isomery nucleic acid molecule and complementary RNA form special double-stranded heterozygote, and be wherein opposite with conventional unit, chain (Gautier C etc., 1987, Nucleic Acids Res 15:6625-6641) parallel to each other.Antisense nucleic acid molecule also can comprise 2 '-O-methyl ribonucleotides (Inoue etc., 1987, Nucleic Acids Res 15:6131-6148) or chimeric RNA-DNA analogue (Inoue etc., 1987, FEBS Lett 215:327-330).

[0330.0.0.0] antisense nucleic acid molecule of the present invention in addition or the antisense nucleic acid molecule that is used for the inventive method be ribozyme also." ribozyme " is meant the catalytic RNA molecule with nuclease, and it can cut the single-chain nucleic acid (as mRNA) that has complementary district with it.The mRNA transcript of polypeptide that ribozyme (for example Haselhoff and Gerlach, 1988, the hammerhead ribozyme of describing among the Nature 334:585-591) can be used for catalytic cutting code book invention polypeptide or is used for the inventive method is to suppress the translation of described mRNA.The proteinic nucleotide sequence that is used for the inventive method based on the nucleotide sequence or the coding of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor, perhaps based on according to the isolating heterologous sequence of the method that the present invention taught, the nucleic acid molecule that can design the polypeptide that uses in code book invention polypeptide or the inventive method has specific ribozyme.For example, can make up the derivative of thermophilas (Tetrahymena) L-19 IVS RNA, wherein the nucleotide sequence complementation among the nucleotide sequence of avtive spot and the coding mRNA that will cut.Consult for example U.S. Patent number 4,987,071 and 5,116 of Cech etc., 742.In addition, use the mRNA of polypeptide to can be used for from the RNA library of molecules, selecting catalytic RNA in code book invention polypeptide or the inventive method with special nuclease.Consult for example Bartel, D. and Szostak, J.W., 1993, Science 261:1411-1418.

[0331.0.0.0] antisense molecule of the present invention also comprises such nucleic acid molecule, and it contains the nucleotide sequence with the regulatory region (for example its promotor and/or enhanser) complementary (for example forming the triple-helix structure that stops the target cell genetic transcription) of the nucleotide sequence of the natural polypeptide that has polypeptide or be used for the inventive method of code book invention (for example peptide sequence of identifying shown in the sequence list or according to methods described herein).Generally see Helene C (1991) Anticancer Drug Res 6 (6): 569-84; (1992) Ann NY Acad Sci 660:27-36 such as Helene C; Maher LJ (1992) Bioassays14 (12): 807-815.

[0332.0.0.0] the invention still further relates to the active double stranded rna molecule of gene product that can reduce or suppress to use in code book invention polypeptide, the inventive method the nucleic acid molecule that uses in gene, nucleic acid molecule of the present invention or the inventive method of polypeptide in addition.

[0333.0.0.0] is by method (the double-stranded RNA interference of double-stranded RNA regulatory gene; ((2000) Plant Mol Biol 43:401-415 such as Matzke MA for example dsRNAi) existingly in animal, yeast, fungi and plant biological body (for example Neurospora, zebra fish, fruit bat, mouse, turbellarian worm, people, trypanosome, morning glory or Arabidopis thaliana) is repeatedly described; (1998) Nature 391:806-811 such as Fire A; WO 99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364).In addition, RNAi is also proved the favourable instrument of inhibition of gene expression in bacterium (for example intestinal bacteria) by [J.Biol.Chem., 2000,275 (34): 26523-26529] such as for example Tchurikov.Fire etc. are with RNAi phenomenon called after " RNA interference ".In the above reference of referring to, technology and method have been described clearly.Efficient gene suppresses also can show (Schweizer P etc., (2000) Plant J 200024:895-903) in (for example, the result who transforms as biological projectile) under the transient expression situation or behind the transient expression.The dsRNAi method is based on the complementary strand of while quiding gene transcript and the efficient inhibition that corresponding chain causes described genetic expression thereof.Phenotype that is produced and the similar mutant closely similar ((1998) Proc Natl Acad Sci USA 95:13959-64 such as Waterhouse PM) that knocks out.

(Gens Dev., 1999,13 (24): 3191-3197) efficient that shows the RNAi method is the function that duplex length, 3 ' distal process go out length and these sequence in outstanding to [0334.0.0.0] Tuschl etc.Work based on Tuschl etc. can give those skilled in the art following guidance: for the result who obtains, should avoid 5 ' and the 3 ' non-translational region of the nucleotide sequence that uses and the zone of close initiator codon, because the adjusting protein binding site is rich in these zones, RNAi sequence and these interactions of regulating between the protein may cause unwanted interaction; The preferred zone of selecting employed mRNA AUG initiator codon downstream 50 to 100nt (=Nucleotide or base); Only the dsRNA (=double-stranded RNA) sequence from exon can be used for present method, because do not have effect from the sequence of intron; G/C content in this zone should be preferably in about 50% greater than 30% and less than 70%; The possible secondary structure of said target mrna is lower to the effect importance of RNAi method.

[0335.0.0.0] confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, 1-5 is capable and/or the expression of the nucleotide sequence of 334-338 shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of one of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.0] term " basic identical " refers to a kind of like this fact, promptly compares with target sequence, and the dsRNA sequence also can have insertion, disappearance and indivedual point mutation, and still causes effective reduction of expression.Preferably, the homology as above definition to (or between complementary strand of " antisense strand " and nucleotide sequence) between the small part sections of inhibition dsRNA " sense strand " and nucleotide sequence of the present invention reaches at least 30%, preferably at least 40%, 50%, 60%, 70% or 80%, extremely preferably at least 90%, most preferably 100%.The length of part sections reaches at least 10 bases, preferred at least 17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 bases, especially preferred at least 40,50,60,70,80 or 90 bases, extremely preferred at least 100,200,300 or 400 bases, most preferably at least 500,600,700,800,900 or more polybase base or at least 1000 or 2000 or more polybase base.In another embodiment preferred of the present invention, the length of part sections reaches 17,18,19,20,21,22,23,24,25,26 or 27 bases, preferred 20,21,22,23,24 or 25 bases.Preferred these short sequences are in animal and plant.Be preferably longer sequence preference between 200 to 800 bases in nonmammalian, preferably in invertebrates, yeast, fungi or bacterium, but also available in plant.Long dsrna is processed into many siRNA (=little/short interfering rna) by for example 3-protein d icer (for double-stranded special Rnase III enzyme) in organism.In addition, " essentially identical " dsRNA also may be defined as can with the nucleotide sequence of portion gene transcript hybridization (for example at 400mM NaCl, 40mMPIPES pH 6.4,1mM EDTA carried out 12 to 16 hours in 50 ℃ or 70 ℃).

[0337.0.0.0] dsRNA can be made up of one or more polymeric ribonucleotide chain.In addition, can exist the two the modification of sugar-phosphate backbone and nucleosides.For example, the phosphodiester bond of natural RNA can be modified in the mode that they comprise at least one nitrogen or sulfur heteroatom.Base can be modified in the mode that for example activity of adenosine deaminase is limited.These and further be modified at the method that hereinafter is used for stabilized antisense rna and describe to some extent.

[0338.0.0.0] dsRNA can prepare by enzyme, or produces by chemosynthesis wholly or in part.

[0339.0.0.0] duplex structure is can be from automatic complementary strand initial or form from two complementary start of chains.When the situation of an automatic complementary strand, there are justice and antisense sequences to connect and form for example hairpin structure by catenation sequence (joint).Preferably, catenation sequence can be intron, and described intron is sheared behind synthetic dsRNA.The nucleotide sequence of coding dsRNA can comprise additional element for example transcription termination signal or polyadenylation signal.If two chains of dsRNA make up in cell or plant, this can take place in many ways.

The formation of [0340.0.0.0] RNA duplex can be initial in extracellular or cell.As shown in WO99/53050, dsRNA also can be by comprising hairpin structure via " joint " connection " justice " and " antisense " chain (for example intron).Preferred self complementary dsRNA structure is because they only need expression construct and the complementary strand of mol ratio such as always comprise.

[0341.0.0.0] uses method hereinafter described, the expression cassette of dsRNA " antisense " or " justice is arranged " chain or dsRNA self complementary strand of preferably will encoding is inserted in the carrier and stable (for example using selective marker) is inserted in the Plant Genome, to guarantee the continuous expression of dsRNA.

[0342.0.0.0] can introduce dsRNA with the amount that makes each at least one copy of cell.Relatively large (for example, each cell at least 5,10,100,500 or 1000 copies) can cause more effective reduction.

[0343.0.0.0] as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.0] is owing to the sequence homology of height between the sequence that derives from multiple biology (for example plant), can reach a conclusion: these protein may be high conservatives in other plant for example, and being expressed in the other plant species of dsRNA that derives from one of open sequence shown in the literary composition or its homologue probably also may have favourable effect.Preferably, consensus sequence shown in the literary composition can be used for being built with the dsRNA molecule of usefulness.

[0345.0.0.0] dsRNA can be in vivo or is external synthetic., the dna sequence dna of coding dsRNA can be imported in the expression cassette for this reason, be at least one Gene Handling element (for example promotor, enhanser, silencer, donor splicing site or acceptor, polyadenylation signal) control down.Suitable useful construct is described hereinafter to some extent.Polyadenylation is optional, also not necessarily will be useful on the element of initial translation.

[0346.0.0.0] dsRNA can be synthetic with chemistry or enzyme process.For this reason, available cell RNA polysaccharase or phage rna polymerase (for example T3, T7 or SP6RNA polysaccharase).Existing describe (WO 97/32016, US 5,593,874, US 5,698,425, US 5,712,135, US 5,789,214, the US 5,804,693) of appropriate method that is used for the RNA vivoexpression., before importing to cell, tissue or organism, can for example extract, the combination of precipitation, electrophoresis, chromatogram or these methods by external chemistry or enzyme process synthetic dsRNA, thereby separate wholly or in part in the reaction mixture.DsRNA can directly import in the cell or (for example importing to the intercellular substance) used in the extracellular.

[0347.0.0.0] RNAi method advantageously only causes the excalation of gene function, and the dosage effect of gene that therefore technician can be studied to work in the purpose biology is also finely tuned the inventive method.It makes those skilled in the art can study the multi-functional of gene in addition.

[0348.0.0.0] yet, plant optimization is with causing that the expression construct that dsRNA expresses carries out stable conversion.Appropriate means is described hereinafter to some extent.

[0349.0.0.0] another embodiment of the present invention also relates to and produces transformed host or host cell (for example eucaryon or prokaryotic cell prokaryocyte, preferred transgenic microorganism, transgenic plant cells or transgenic plant tissue or transgenic plant) method, it comprises importing nucleic acid construct of the present invention, carrier of the present invention or nucleic acid molecule of the present invention in plant, vegetable cell or plant tissue.

[0350.0.0.0] another embodiment of the present invention also relates to instantaneous generation host or host cell (for example eucaryon or prokaryotic cell prokaryocyte, preferred transgenic microorganism, transgenic plant cells or transgenic plant tissue or transgenic plant) method, it comprises importing in plant, vegetable cell or plant tissue describes nucleic acid molecule or the nucleic acid molecule of the present invention that is included in the nucleic acid construct in nucleic acid construct of the present invention, carrier of the present invention or the literary composition, the nucleic acid molecule of Dao Ruing, nucleic acid construct and/or carrier unconformability are advanced in the genome of host or host cell thus.Therefore in host's breeding, transformant is unsettled with regard to the nucleic acid molecule, nucleic acid construct and/or the carrier that import.

[0351.0.0.0] if genetically modified organism is the plant form, then also can be regarded as growth with the vegetable cell that produces each fine chemicals, plant tissue, plant organ for example root, bud, stem, flower, piece root or leaf in the methods of the invention, or whole strain plant.

[0352.0.0.0] growth is interpreted as for example cultivating transgenic plant cells, plant tissue or plant organ on nutritional medium, or cultivates whole strain plant on (for example water culture thing, potted plant culture) or soil on the matrix.

[0353.0.0.0] is in another favourable embodiment of present method, can (see Falciatore etc. single celled vegetable cell (for example algae), 1999, Marine Biotechnology 1 (3): 239-251 and the reference wherein quoted) or the vegetable cell of higher plant (for example spermatophyte for example crop) in the express nucleic acid molecule.The example of plant expression vector comprise describe in detail in the literary composition or Becker, D.[(1992) Plant Mol.Biol.20:1195-1197] and Bevan, M.W.[(1984), Nucl.Acids Res.12:8711-8721; Vectors for Gene Transfer in Higher Plants; In:Transgenic Plants, volume 1, Engineering and Utilization, Kung and R.Wu compile, Academic Press, 1993, pp.15-38] in those.Hellens is seen in the commentary of binary vector and uses thereof, R.[(2000), and Trends in Plant Science, 5 (10): 446-451.

[0354.0.0.0] can introduce protokaryon or eukaryotic cell with carrier DNA by routine conversion or rotaring dyeing technology.Term " conversion " and " transfection " comprise combination and transduction, and be intended to comprise the multiple existing method that is used for introducing exogenous nucleic acid molecule (for example DNA) to host cell as used in the literary composition of the present invention, comprise transfer, electroporation or the particle bombardment of transfection, the lipofection of transfection, the PEG mediation of coprecipitation of calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-dextran, natural competence, chemical process mediation.Be used to transform or change the sense host cell and comprise that the proper method of vegetable cell is found in (" Molecular Cloning:A Laboratory Manual. " such as Sambrook, the 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 1989) and other laboratory manuals as " Methods inMolecular Biology ", 1995, the 44 volumes, Agrobacterium protocols, Gartland and Davey compile, Humana Press, Totowa, New Jersey.

[0355.0.0.0] use is above-mentioned to be used to transform and the instantaneous or stable conversion plant from the method for plant tissue or vegetable cell regeneration plant.Suitable method is by " biological projectile " method (being also referred to as microprojectile bombardment methods), the electroporation of the protoplast transformation of polyoxyethylene glycol inductive DNA picked-up, use particle gun, hatches dried embryo, microinjection and agriculture bacillus mediated transgenosis in dna solution.Aforesaid method is described in for example B.Jenes etc., " Transgenic Plants ", the first roll, Engineeringand Utilization, S.D.Kung and R.Wu compile, the Techniques for Gene Transfer of Academic Press (1993) 128-143 and Potrykus Annu.Rev.Plant Physiol.PlantMolec.Biol.42 (1991) 205-225.Treating that expression construct is preferably cloned into is fit to transform in the carrier of agrobacterium tumefaciens, for example pBin19 (Bevan etc., Nucl.Acids Res.12 (1984) 8711).Subsequent transformation the Agrobacterium of this carrier can be used for transforming particularly crop plants of plant by known way, as tobacco plant, for example the leaf or the leaf segment of scratching is immersed in the Agrobacterium solution, in suitable medium, cultivate subsequently.The Plant Transformation that relies on agrobacterium tumefaciens is by for example

With Willmitzer at Nucl.Acid Res. (1988) 16, describe in 9877, or be known in F.F.White in addition, " Transgenic Plants ", the 1st volume, Engineering and Utilization, S.D.Kung and R.Wu compile, Academic Press, 1993, " the Vectors forGene Transfer in Higher Plants " of 15-38 page or leaf.

[0356.0.0.0] for selecting successfully to be transferred to according to the present invention nucleic acid molecule of the present invention, carrier or the nucleic acid construct of host cell, it is favourable using the marker gene of above having described in detail.Known when stable or integration,temporal advances vegetable cell at nucleic acid molecule, have only a few cell picked-up foreign DNA and be integrated into its genome (if necessary), this depends on the expression vector of use and the rotaring dyeing technology of use.For identifying and select these intasomies, the gene of the selective marker of will encoding usually (as indicated above, for example antibiotics resistance) is introduced host cell with goal gene.Preferred selective marker comprises the mark of giving the resistance of weedicide such as glyphosate or careless fourth phosphine in the plant.The mark (as beta-galactosidase enzymes, ura3 or ilv2) that is labeled as for example encode sugar or amino acid biosynthetic pathway genes involved that other are suitable.Coding is suitable as the mark of luciferase, gfp or other fluorogenes equally.These marks and mark mentioned above can be used for mutant, and these genes in described mutant are not owing to for example had function by ordinary method by disappearance.In addition, the nucleic acid molecule of coding selective marker can be introduced host living beings with the nucleic acid molecule of code book invention polypeptide or polypeptide that present method is used in identical carrier or in independent carrier.Can be for example by having selected to identify stable transfection the cell of the nucleic acid of introducing (for example having integrated the cell survival of selective marker and other necrocytosiss).

[0357.0.0.0] in case nucleic acid successfully introduce, genetically modified host cell just no longer needs or does not wish underlined gene (usually particularly to the gene of microbiotic and Herbicid resistant), advantageously uses the technology that can remove or excise these marker gene so the present invention introduces the method for nucleic acid.Known a kind of these class methods are cotransformation.Co-transformation method of particle uses two carriers to transform simultaneously, a carrying nucleic acid of the present invention, second bearing mark gene.Most transformant (up to 40% or higher transformant) accept or (under the plant situation) contains whole two carriers.For the situation of Agrobacterium-mediated Transformation, common receiving portion fractional bearer one both sides of transformant are the sequence of T-DNA, and it is representing expression cassette usually.From transform plant, remove marker gene by hybridization subsequently.In other method, marker gene is integrated in the transposon, and is used from conversion (being called the Ac/Ds technology) with required nucleic acid one.Transformant can be hybridized with the transposase source, perhaps with giving the instantaneous or stable conversion transformant of nucleic acid construct that transposase is expressed.(about 10%) in some cases, transposon is jumped out the host cell gene group and is lost after successfully transforming.Under the other situation, transposon jumps to different positions.In these cases, must eliminate marker gene by hybridization.In microbiology, developed and made these incidents of detection become possibility or easier method.Another advantageous method depends on known recombination system, and its superiority is eliminated for saving hybridization.The best system of the type is the system that is called Cre/lox.Cre1 is a recombinase of removing sequence between the loxP sequence.If marker gene is incorporated between the loxP sequence, it is removed by the recombinase expression after successfully transforming.Other recombination systems are HIN/HIX, FLP/FRT and REP/STB system (Tribble etc., J.Biol.Chem., 275,2000:22255-22267; Velmurugan etc., J.Cell Biol., 149,2000:553-566).Nucleotide sequence of the present invention can be advanced in the Plant Genome with site-specific integration.These methods also can be used for microorganism such as yeast, fungi and bacterium naturally.

The Agrobacterium that [0358.0.0.0] transformed expression vector of the present invention can (for example be immersed in the leaf or the leaf segment of scratching in the Agrobacterium solution by known way own equally, in suitable medium, cultivate subsequently) be used for Plant Transformation, described plant such as test plant such as Arabidopis thaliana or crop plants, as cereal, corn, oat, rye, barley, wheat, soybean, rice, cotton, sugar beet, rape, Sunflower Receptacle, flax, hemp, potato, tobacco, tomato, Radix Dauci Sativae, big capsicums (bellpepper), rape, cassava (tapioca), cassava (cassava), Pueraria lobota (arrow root), Flower of Aztec Marigold, alfalfa, lettuce and various trees, nut and grape vine (grapevine), oil-containing crop plants particularly is as soybean, peanut, the Viscotrol C plant, Sunflower Receptacle, corn, cotton, flax, rape, coconut, palm, safflower (Carthamus tinctorius) or cocoa beans.

[0359.0.0.0] can also transform plant meristematic cell and particularly growth and be those cells of gamete except transformant cell (it must be regenerated as complete plant).In this case, the gamete of conversion carries out natural phant growth generation transgenic plant.Therefore, for example handle the seed of Arabidopis thaliana with Agrobacterium, and transformed from certain proportion and therefore obtain seed (Feldman, KA and Marks MD (1987) .Mol Gen Genet 208:274-289 for genetically modified developmental plant; Feldmann K (1992) .C Koncz, N-H Chua and J Shell compile, " Methods inArabidopsis Research ", Word Scientific, Singapore, 274-289 page or leaf).Alternative method is hatched based on the repeated removal inflorescence with the Agrobacterium that transforms and the excision site at lotus throne center, and (Chang (1994) .Plant J.5:551-558 for the seed that can obtain transforming of time point afterwards thus; Katavic (1994) .Mol Gen Genet, 245:363-370).Yet special effective means is the vacuum infiltration method and improves one's methods, as " flower-dipping method (floral dip) ".Under the vacuum infiltration situation of Arabidopis thaliana, with the complete plant (Bechthold under the agrobacterium suspension processing low pressure, N (1993) .C R Acad Sci Paris Life Sci, 316:1194-1199), and in " flower-dipping method " situation, the agrobacterium suspension that the flower tissue of growth is handled with tensio-active agent is hatched (Clough, SJ and Bent in short-term, AF (1998) .The Plant J.16,735-743).In two kinds of situations, can gather in the crops a certain proportion of transgenic seed,, these seeds and non-transgenic seed zone be separated by under aforementioned selection condition, cultivating.In addition, the stable conversion of plastid is favourable, because plastid is a matrilinear inheritance in most crops, this has reduced or eliminated by the transgenosis drift of pollen dangerous.Generally by Klaus etc. at Nature Biotechnology 2004,22 (2), graphic technique is finished the conversion of chloroplast gene group among the 225-229.In brief, sequence to be transformed and the selectable marker gene between chloroplast gene group homologous flanking sequence are cloned.These homologous flanking sequence instruct locus specificity to be integrated in the plastom(e).The plastid of having described many different plant species transforms, and is summarized in Bock (2001) Transgenic plastids in basic researchand plant biotechnology.J Mol Biol.2001 Sep 21; 312 (3): 425-38 or Maliga, P (2003) Progress towards commercialization of plastidtransformation technology.Trends Biotechnol.21,20-28.Reported the other biological technical progress of unmarked plastid transformant form recently, described transformant can produce by the instantaneous marker gene of integrating altogether (Klaus etc., 2004, Nature Biotechnology 22 (2), 225-229).

[0360.0.0.0] genetically modified vegetable cell can be regenerated by all methods that those skilled in the art are familiar with.Appropriate means is found in aforementioned S.D.Kung and R.Wu, Potrykus or Document with Willmitzer.

[0361.0.0.0] therefore the invention still further relates to the vegetable cell that contains nucleic acid construct of the present invention, nucleic acid molecule of the present invention or carrier of the present invention.

[0362.0.0.0] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or each fine chemicals of its part increases) carry out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide or be used for the inventive method polypeptide (for example as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, polypeptide of the present invention or be used for the polypeptide of the inventive method or the cytoactive of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor improves, for example since in cell or biological or its part the expression of research object of the present invention or specific activity improve.In one embodiment, have as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part as Table II the 3rd row, 1-5 is capable and/or 334-338 capable shown in polypeptide (for example, have as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide of sequence) activity improve.Example and the inventive method are described in above.

[0363.0.0.0] " genetically modified " (for example about the nucleic acid molecule, nucleic acid construct or the carrier that contain described nucleic acid molecule or the biology that transforms with described nucleic acid molecule, nucleic acid construct or carrier) refers to all experimenters of producing by recombination method, wherein

A) nucleotide sequence, or

B) the Genetic Control sequence that effectively is connected with nucleotide sequence, as promotor, or

C) (a) and (b)

Be not arranged in its natural genotypic environment or modify by recombination method, the example of modification is replacement, interpolation, disappearance, inversion or the insertion of one or more nucleotide residues.Natural genotypic environment refer to originate in the biology natural dyeing position point or be present in the genomic library.In the situation of genomic library, preferred (to small part) kept the natural genotypic environment of nucleotide sequence.Environment is at least in nucleotide sequence one side, and has the sequence that length is at least 50bp, preferably 500bp, especially preferably 1000bp, extremely preferred 5000bp at least at least at least.

[0364.0.0.0] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---combination of for example naturally occurring polypeptide promotor of the present invention and respective egg white matter encoding sequence---becomes transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.0] in addition can also transformed plant cells, plant tissue or plant, thereby (mistake) expresses other enzymes and protein, and described enzyme and protein expression are supported the increase of each fine chemicals.

[0366.0.0.0] yet, transgenosis means that also nucleic acid of the present invention is arranged in them on the natural place of biological gene group, but sequence is compared with native sequences and is modified and/or the adjusting sequence of native sequences is modified.Preferably, transgenosis/reorganization is interpreted as that also the transcribed nucleic acid that uses in the inventive method takes place in genomic non-natural position, that is to say that expression of nucleic acids is a homologous, or preferably allogenic.This expresses instantaneous, or stable integration advances the expression of genomic sequence.

The term " transgenic plant " that uses among [0367.0.0.0] the present invention also relates to the offspring of transgenic plant, for example T ₁, T ₂, T ₃With follow-up plant generation or BC ₁, BC ₂, BC ₃With the follow-up plant generation.Therefore, transgenic plant of the present invention can be cultivated also selfing or hybridize to obtain more transgenic plant of the present invention with other individualities.Also can obtain transgenic plant by the vegetative propagation transgenic plant cells.The invention still further relates to can be from the transgenic plant material of transgenic plant population of the present invention.This material comprises certain tissue, organ and the plant part of vegetable cell and its all manifestation, for example the material of seed, leaf, flower pesticide, fiber, stem tuber, root, root hair, stem, embryo, callus, cotyledon, petiole, results, plant tissue, breeding tissue and from real transgenic plant also/or can be used for producing the cell cultures of transgenic plant.

Any conversion plant that [0368.0.0.0] obtains according to the present invention can be used in traditional breeding system or in the external plant propagation to produce the conversion plant that more has identical characteristics and/or to can be used in other mutation of identical or relevant species, introducing identical characteristics.This type of plant also is a part of the present invention.In heredity, also comprise identical characteristic and be part of the present invention from the seed that transforms the plant acquisition.As previously mentioned, the present invention is applicable to any plant and the crop that can be well known by persons skilled in the art any method for transformation transforms in principle.

[0369.0.0.0] in particularly preferred embodiments, biology of the present invention, host cell, vegetable cell, plant, microorganism or plant tissue are genetically modified.

[0370.0.0.0] therefore, the genetically modified organism of at least a nucleic acid molecule of the present invention, nucleic acid construct or carrier and cell, cell culture, tissue, the reproductive material of (for example the plant tissue under the plant biological situation, as leaf, root etc.) or or complete plant biological have partly been the present invention relates to transform from these.Term " reorganization (host) " and " transgenosis (host) " are used interchangeably in context.Certainly these terms not only relate to described host living beings or target cell, and relate to the offspring or the potential offspring of these biologies or cell.Because sudden change or environmental influence may form some modification in the successive generation, these offsprings are not necessarily identical with parental cell, but still are included in the scope that this paper uses term.

[0371.0.0.0] is used for the inventive method or can synthesizes the eucaryon or the prokaryotic organism of each fine chemicals as host's suitable biology for all.The biology that uses as the host is microorganism (for example bacterium, fungi, yeast or algae), non-human animal or plant (for example dicotyledonous or monocotyledons).

[0372.0.0.0] in principle, all plants can be used as host living beings, particularly above-mentioned as the source organism plant.Preferred transgenic plant are for for example being selected from Aceraceae, Anacardiaceae, umbelliferae, composite family, brassicaceae, Cactaceae, Curcurbitaceae, Euphorbiaceae, pulse family (Fabaceae), Malvaceae, Nymphaeceae, papaveracease, the Rosaceae, Salicaceae, Solanaceae, Palmae (Arecaceae), Bromelia family, Cyperaceae, Iridaceae, Liliaceae, the orchid family, Gentianaceae, Labiatae, Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae, scrophulariaceae, Caryophyllaceae, Ericaceae, polygonaceae, Violaceae, rush family or plant gramineous, and be preferably selected from umbelliferae, composite family, brassicaceae, Curcurbitaceae, pulse family (Fabaceae), papaveracease, the Rosaceae, Solanaceae, the plant of Liliaceae or Gramineae (Poaceae).Crop plants preferably, for example advantageously be selected from peanut (peanut), rape (oilseed rape), rape (canola), Sunflower Receptacle (sunflower), safflower (safflower), olive (olive), sesame (sesame), fibert (hazelnut), apricot (almond), avocado (avocado), bay, pumpkin (pumpkin/squash), linseed oil (linseed), soybean (soya), pistachio (pistachio), Borrago officinalis (borage), corn (maize), wheat (wheat), rye (rye), oat (oats), jowar (sorghum) and millet (millet), triticale (triticale), rice (rice), barley (barley), cassava (cassava), potato (potato), sugar beet (sugarbeet), eggplant (egg plant), alfalfa (alfalfa) and perennial herb and forage plant, coconut oil (oil palm), vegetables (rape, root vegetable, tuberous vegetable, beanpod class vegetables, the fruits vegetables, onion class vegetables, leafy vegetable and stem vegetables), buckwheat (buckwheat), jerusalem artichoke (Jerusalem artichoke), broad bean (broad bean), common vetch (vetches); root of Szemao crotalaria (lentil); string bean (dwarf bean); lupine (lupin); the plant of trifolium (clover) and alfalfa (lucerne), that mentions is just wherein a part of.

[0373.0.0.0] preferred vegetable cell, plant organ, plant tissue or plant part derive from the plant section that is mentioned in the biology of source, preferably belong to from above-mentioned plant, more preferably from above-mentioned plant species.

[0374.0.0.0] contains in the methods of the invention the transgenic plant of institute's synthesizing amino acid and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the amino acid that produces in the inventive method also can be from plant with total free aminoacids or be incorporated into isolated in form in the protein.The amino acid that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or the ion-exchange chromatography of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.0] the present invention relates to produce method of microorganism in another embodiment, comprises nucleic acid construct of the present invention or carrier of the present invention or nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor importing microorganism or its part.

[0376.0.0.0] the invention still further relates to the transgenic microorganism that comprises nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention or carrier of the present invention in another embodiment.The microorganism that is fit to is described in the biology of the source of this paper, the preferred particularly above-mentioned bacterial strain that is suitable for producing fine chemicals.

[0377.0.0.0] therefore the invention still further relates to the method for amino-acids composition that separation produces or each fine chemicals that is produced.

[0378.0.0.0] can separate and be higher than 50% by weight whereby, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than 90% the fine chemicals that produces in the method.If suitable, the fine chemicals that produces can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.0.0] in one embodiment, lipid acid is fine chemicals.

The suitable synthetic parent material of the amino acid that [0380.0.0.0] obtains in the method for the invention as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises amino-acids composition that separation produces or fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is amino acid or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine that the inventive method produces.

[0381.0.0.0] can use all microorganisms (those that mention in the biology of particularly above-mentioned source) as host living beings in principle.Advantageously, use transgenic microorganism in the methods of the invention, fungi for example, as Claviceps or aspergillus, perhaps gram-positive microorganism belongs to or genus arthrobacter as bacillus, Corynebacterium, micrococcus sp, brevibacterium sp, Rhod, Nocardia, butter bacillus, perhaps Gram-negative bacteria, as Escherichia, Flavobacterium or salmonella, perhaps yeast is as Rhodotorula, Chinese Sen Shi yeast belong or mycocandida.Particularly advantageous biology is selected from Corynebacterium, brevibacterium sp, Escherichia, bacillus, Rhodotorula, Chinese Sen Shi yeast belong, mycocandida, Claviceps or Flavobacterium.Particularly advantageously the microorganism of using in the methods of the invention is selected from unusual debaryomyces hansenii, Candida utilis, ergot, Bacillus circulans, subtilis, bacillus certain (Bacillus sp.), Brevibacteriumalbidum, Brevibacterium album, Brevibacterium cerinum, brevibacterium flavum, Brevibacterium glutamigenes, brevibacterium iodinum, Brevibacteriumketoglutamicum, brevibacterium lactofermentum, extension brevibacterium, Brevibacteriumroseum, Brevibacterium saccharolyticum, brevibacterium sp certain (Brevibacterium sp.), Corynebacterium acctoacidophlum, Corynebacterium acetoglutamicum, produce ammonia rod bacillus, Corynebacterium glutamicum (=micrococcus glutamicus), corynebacterium melassecola, Corynebacterium certain (Corynebacterium sp.) or intestinal bacteria, particularly e. coli k12 and described bacterial strain thereof.

When [0382.0.0.0] was microorganism when host living beings, method of the present invention advantageously between 0 ℃ and 95 ℃, between preferred 10 ℃ and 85 ℃, between 15 ℃ and 75 ℃, was extremely preferably finished between 15 ℃ and 45 ℃.PH advantageously remains between the pH 4 to 12 therebetween, preferably between pH 6 to 9, between pH 7 to 8.Can criticize formula, half batch of formula or continue to carry out the inventive method.Known cultural method general introduction is found in Chmiel (" Bioproze β technik 1.Einf ü hrung in die Bioverfahrenstechnik " (Gustav Fischer Verlag, Stuttgart, 1991) in the textbook) or Storhas (" Bioreaktoren und periphereEinrichtungen " (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) in the textbook).Substratum to be used must satisfy the needs of bacterial strain separately by rights.The substratum explanation that is used for multiple microorganism appears in " Manual of Methods for GeneralBacteriology " handbook of American Society for Bacteriology (Washington D.C., USA, 1981).As mentioned above, can be used for these substratum of the present invention and comprise one or more carbon sources, nitrogenous source, inorganic salt, VITAMIN and/or trace element usually.Preferred carbon source is sugar, for example monose, disaccharides or polysaccharide.The example of extraordinary carbon source is glucose, fructose, seminose, semi-lactosi, ribose, sorbose, ribulose lactose, maltose, sucrose, raffinose, starch or Mierocrystalline cellulose.Also can pass through complex chemical compound (for example byproduct of molasses or other sugar refinings) and in substratum, add sugar.It also is favourable adding the several kinds of carbon source mixture.Other possible carbon sources are oil ﹠ fat, for example soybean oil, sunflower oil, peanut oil and/or coconut fat; Lipid acid is as Palmiticacid, stearic acid and/or linolic acid; Alcohol and/or polyvalent alcohol, for example glycerine, methyl alcohol and/or ethanol; And/or organic acid, for example acetate and/or lactic acid.Nitrogenous source is generally the organic or inorganic nitrogen compound or comprises the material of these compounds.The example of nitrogenous source comprises ammonia or ammonium salt liquid or gaseous form, for example ammonium sulfate, ammonium chloride, ammonium phosphate, volatile salt or ammonium nitrate, nitrate, urea, amino acid or compound nitrogen source, for example corn steep liquor, soyflour, soy-protein, yeast extract, meat extract and other.Nitrogenous source can use separately or use with mixture.The inorganic salt compound that can exist in the substratum comprises muriate, phosphoric acid salt or the vitriol of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0383.0.0.0] is in order to prepare the sulfur-bearing fine chemicals, each fine chemicals (for example sulfur-containing amino acid) particularly, can use inorganic sulfocompound for example vitriol, sulphite, hyposulfite, tetrathionate, thiosulphate, sulfide or other organosulfur compounds (for example mercaptan and sulfhydryl compound) as the sulphur source.

[0384.0.0.0] can use phosphoric acid, potassium primary phosphate or dipotassium hydrogen phosphate or the corresponding sodium salt that contains as the phosphorus source.Can in substratum, add sequestrant to keep the solution metal ion.Specially suitable sequestrant comprises dihydroxy phenol (dihydroxyphenols) (for example pyrocatechol) or former benzoic acid (protocatechuate) or organic acid (for example citric acid).The fermention medium that is used for culturing micro-organisms that the present invention uses also comprises other somatomedins usually, and for example VITAMIN or positive growth factor comprise biological example element, riboflavin, thiamines, folic acid, nicotinic acid, pantothenic acid and pyridoxol.Somatomedin and salt is usually from the complex medium composition, for example yeast extract, molasses, corn steep liquor etc.Can in substratum, add suitable precursor in addition.The substratum mixture is cut into branch really and greatly depends on concrete experiment and select separately for each specific case.About the visible textbook of the information of medium optimization " Applied Microbiol.Physiology, A Practical Approach " (P.M.Rhodes compiles, P.F.Stanbury, IRL Press (1997) 53-73 page or leaf, ISBN 0 19,963,577 3).Growth medium also can be available from commercial supplier (for example Standard 1 (Merek) or BHI (brain heart leach liquor, DIFCO) etc.).All medium components can be sterilized by heating (1.5 crust and 121 ℃ continue 20 minutes) or sterilising filtration.Composition can be together or (if desired) sterilization separately.All medium components can exist when cultivation is initial or optional lasting the adding or adding in batches.Culture temperature usually between 15 ℃ and 45 ℃, preferred 25 ℃ to 40 ℃, and can in experiment, keep constant or change.The pH of substratum should be in 5 to 8.5 scopes, and preferred about 7.Can be in cultivation by adding the pH that the control of basic cpd (for example sodium hydroxide, potassium hydroxide, ammonia or ammoniacal liquor) or acidic cpd (for example phosphoric acid or sulfuric acid) is cultivated.Can use defoamer (for example fatty acid polyglycol ester) control foam.Can be by in substratum, adding the stability that material suitable, that have selection effect (for example microbiotic) keeps plasmid.Aerobic condition keeps by introducing oxygen or oxygen-containing gas mixture (for example air) in culture.Culture temperature is usually from 20 ℃ to 45 ℃, and preferably from 25 ℃ to 40 ℃.Cultivation lasts till that the purpose product forms with maximum.This target realized in 10 hours to 160 hours usually.

[0385.0.0.0] particularly contains for example fermented liquid of L-methionine(Met), L-Threonine and/or L-Methionin with what this kind approach obtained, and dry matter content accounts for 7.5 to 25% of weight usually.At least when finishing, particularly passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means the concentration that can utilize sugar in the fermention medium during this period of time remain on or be reduced to 〉=0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant) or these methods or partly from fermented liquid, shift out or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.0.0] yet, can also be further purified the amino acid of generation.For this reason, under all or part of situation that stays in chromatography resin of purpose product or impurity, will comprise component chromatogram on appropriate resin of product.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.The product of purifying can pass through to filter or ultrafiltration and concentration, and is kept under the temperature of product stability maximum.

[0387.0.0.0] can determine the characteristic and the purity of institute's separating compound by prior art.These methods comprise high performance liquid chromatography (HPLC), spectrographic technique, mass spectrum (MS), staining, thin-layer chromatography, NIRS, enzyme analysis or microbiological analysis.These analytical methods are summarized in people such as Patek, (1994) Appl.Environ.Microbiol.60:133-140; People such as Malakhova, (1996) Biotekhnologiya 11 27-32; With people such as Schmidt, (1998) Bioprocess Engineer.19:67-70; Ulmann ' s Encyclopedia of Industrial Chemistry (1996) rolls up A27, VCH:Weinheim, 89-90 page or leaf, 521-540 page or leaf, 540-547 page or leaf, 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas ofBiochemistry and Molecular Biology ", John Wiley and Sons; Fallon, people such as A., (1987) Applications of HPLC in Biochemistry in:LaboratoryTechniques in Biochemistry and Molecular Biology, volume 17.

[0388.0.0.0] on the other hand, the invention still further relates to part gathered in the crops and the reproductive material of transgenic plant of the present invention, itself or comprise the transgenic plant cells of expressing nucleic acid molecule of the present invention, the cell of cytoactive that perhaps comprises the raising of the polypeptide that shows polypeptide of the present invention or be used for the inventive method, for example described protein expression level of Ti Gaoing or higher activity.

[0389.0.0.0] can gather in the crops any available part that part generally can be plant, for example flower, pollen, seedling, stem tuber, leaf, stem, fruit, seed, root etc.Reproductive material comprises for example seed, fruit, cutting (cutting), seedling, stem tuber, root stock etc.Can gather in the crops part or reproductive material preferred seed, fruit, seedling or stem tuber.

[0390.0.0.0] the invention still further relates to genetically modified organism of the present invention and cell, cell culture, from their part for example as above to biological described of transgenic plant, leaf etc. and the rotaring gene breeding material purposes that is used to produce food or feed, medicine or fine chemicals such as seed or fruit for example as mentioned above.

[0391.0.0.0] therefore in another embodiment, the present invention relates to nucleic acid molecule of the present invention, biology (for example microorganism, plant, vegetable cell or plant tissue), carrier or polypeptide and be used for make producing lipid acid, carotenoid, isoprenoid, VITAMIN, lipid, wax ester, (many) sugar and/or poly hydroxy fatty acid, and/or its meta-bolites purposes of cell, tissue and/or the plant of steroid hormone, cholesterol, prostaglandin(PG), triacylglycerol, bile acide and/or ketoboidies particularly.A large amount of mechanism influences lipid acid, carotenoid, isoprenoid, VITAMIN, wax ester, lipid, (many) sugar and/or poly hydroxy fatty acid, and/or its meta-bolites particularly steroid hormone, cholesterol, triacylglycerol, prostaglandin(PG), bile acide and/or ketoboidies or other above-mentioned output, generation and/or generation efficient that changes proteinic fine chemicals in conjunction with this class.Under the plant situation, by for example increasing the expression of acetyl-CoA in the cell, might increase the quantity of the described compound of generation, thereby allow more easily to collect and purifying or more effective distribution (under the situation of factory), described acetyl-CoA is for example lipid acid, carotenoid, isoprenoid, VITAMIN, lipid, (many) sugar, wax ester and/or poly hydroxy fatty acid and/or its meta-bolites bases that produce of prostaglandin(PG), steroid hormone, cholesterol, triacylglycerol, bile acide and/or ketoboidies particularly of many products.In addition, the increase of the intermediate compound quantity of one or more described meta-bolitess, cofactor, precursor molecule and suitable biosynthetic pathway may be essential.Therefore, participate in the nutrient substance for example quantity and/or the activity of the transferrin of carbon source (i.e. sugar), nitrogenous source (being amino acid, ammonium salt), phosphoric acid salt and sulphur input by increasing, may be owing to the generation of having removed any allowance of nutrients restriction improvement acetyl-CoA and the above-mentioned meta-bolites thereof in the biosynthetic process.Particularly, can increase described compound (for example lipid acid, carotenoid, isoprenoid, VITAMIN, wax ester, lipid, (many) sugar and/or poly hydroxy fatty acid, and/or its meta-bolites particularly steroid hormone, cholesterol, prostaglandin(PG), triacylglycerol, bile acide and/or ketoboidies molecule) in plant output, generation and/or produce efficient.

[0392.0.0.0] other preferably reorganization in host living beings produces the method for medicine or fine chemicals, wherein host living beings transforms with one of above-mentioned nucleic acid construct (comprising one or more coding purpose fine chemicals or the biosynthetic structure gene of catalysis purpose fine chemicals), cultivates transformed host cells and separate the purpose chemical from substratum.This method can be widely used in fine chemicals, for example enzyme, VITAMIN, amino acid, sugar, lipid acid and natural and mixed condiments, spices and dyestuff or comprise their mixture.Particularly preferably be other amino acid of extra generation, tocopherol and tocotrienols and carotenoid or comprise the mixture of described compound.It is biological and reclaim product by method known to those skilled in the art from host living beings or substratum to cultivate host transformed, or with biology self as food or fodder additives.The generation of medicine (for example antibody or vaccine) is described in Hood EE, Jilka JM.Curr Opin Biotechnol.1999 Aug; 10 (4): 382-6; Ma JK, Vine ND.Curr Top Microbiol Immunol.1999; 236:275-92.

[0393.0.0.0] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 1-5 is capable and/or 334-338 is capable, preferred Table I B the 5th or 7 row, 1-5 is capable and/or the nucleic acid molecule of 334-338 shown in capable hybridized under lax stringent condition nucleic acid molecule, and randomly separates full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.0] lax hybridization conditions is: after the standard crossover operation, can under the moderate stringent condition, carry out washing step low, with respect to strict wash conditions (for example 60 ℃-68 ℃, 0.1%SDS), normally used wash conditions is the salt condition between 40 ℃-55 ℃, 0.1%SDS, 2xSSC and 0.2x SSC.Other stringent hybridization condition examples are found in above-listed reference.Usually washing step repeats with strict degree and the length that increases, up to detecting useful signal/noise ratio, this depends on many factors, as target (for example its purity, GC content, size etc.), probe (for example its length, be rna probe or dna probe), salt condition, washing or hybridization temperature, washing or hybridization time etc.

[0395.0.0.0] the present invention relates to identify the method for giving the gene product that each fine chemicals generation increases in the cell in another embodiment, and it comprises the following steps:

(a) nucleic acid molecule of identification of organism; Described biology may comprise candidate gene (giving the gene product that each fine chemicals increases after being coded in expression), itself and nucleic acid molecule of the present invention have at least 20%, preferred 25%, more preferably 30%, preferred more especially 35%, 40% or 50%, preferred more especially 60%, 70% or 80%, most preferably 90% or 95% or higher homology, for example by in database, carrying out the homology search;

(b) the candidate nucleic acid molecule is introduced the host cell that is suitable for producing each compound that becomes more meticulous, in preferred plant cell or the microorganism;

(c) in host cell, express the nucleic acid molecule of identifying;

(d) level of each fine chemicals in the mensuration host cell; With

(e) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

At last, can also identify the gene product of giving each fine chemicals product increase according to the same or analogous 3D structure in the step (a) and by aforesaid method.

The nucleic acid molecule that [0396.0.0.0] can use evaluation then is to produce each fine chemicals with the same method of nucleic acid molecule of the present invention.Therefore, in one embodiment, the present invention relates to produce the method for each fine chemicals, comprise that (a) identifies nucleic acid molecule and reclaim free or bonded fine chemicals from biology to (e) according to abovementioned steps (a) to (f) or (a), compare with wild-type, this biology has the cytoactive of institute's isolated nucleic acid molecule coded polypeptide of raising.

[0397.0.0.0] in one embodiment, the present invention relates to identify the method that stimulates the compound that each fine chemicals produces in the described plant that it comprises in addition:

A) cell that will express polypeptide of the present invention or its mRNA contacts under cell culture condition with candidate compound;

B) measure the raising that described polypeptide or described mRNA express;

The standard reaction that produces when c) relatively expression level is with no described candidate compound; Thus, the expression that improves than standard illustrates that this compound stimulates the generation of each fine chemicals.

[0398.0.0.0] in addition, in one embodiment, the present invention relates to screen the method for (for example giving the polypeptide that each fine chemicals increases in biology or its part after improving activity) active agonist of the polypeptide that uses in polypeptide of the present invention or the inventive method or antagonist, it comprises:

(a) cell, tissue, plant or the microorganism that will express polypeptide of the present invention and candidate compound or comprise under the condition of sample expression of polypeptides that uses in allowing polypeptide of the present invention or the inventive method of multiple compound contacts;

(b) each fine chemicals level or the expression of polypeptides level in the substratum of mensuration cell, tissue, plant or microorganism or cultivation or maintenance cell, tissue, plant or microorganism; With

(c) each fine chemicals level by measuring, or in polypeptide of the present invention or the inventive method the expression of polypeptides level of using and no described candidate compound or each fine chemicals or the comparison of expression of polypeptides level standard of measuring when comprising the sample of described multiple compound, identify agonist or antagonist; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

[0399.0.0.0] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving fine chemicals generation raising under these conditions.

[0399.1.0.0] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing whether this effect depends on as Table II the 5th or 7 row, 1-5 is capable and/or the polypeptide of 334-338 shown in capable or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.0] described compound can be chemosynthesis or microorganisms and/or be contained in the sample (as cell extract) from for example plant, animal or microorganism (as pathogenic agent).In addition, described compound may be known in the art, can not suppress or activate polypeptide of the present invention but still do not understand it so far.Reaction mixture can be not celliferous extract or can contain the cell or tissue culture.The inventive method is suitable be set to it be known to those skilled in the art that and for example general description in Alberts etc., " Molecular Biology of the Cell ", the third edition (1994), particularly 17 chapters.This compound can for example add in reaction mixture, the substratum, be injected in the cell or spray on the plant.

[0401.0.0.0] is if identified the sample that contains compound in the methods of the invention, then can contain separating compound in the primary sample that can activate or improve the compound of each fine chemicals content biology or its part from being accredited as, perhaps can further segment primary sample (if for example containing multiple different compound) reducing the quantity of different substances in each sample, and with the part repetition methods of primary sample.The complicacy that depends on sample can be carried out step several times mentioned above, and preferably the sample of identifying until the inventive method only contains limited several or only contain a kind of material.Preferred described sample contains chemistry and/or the similar material of physical property, and most preferably described material is identical.Preferably, to be fit to the further preparation of the form method compounds identified or derivatives thereof mentioned above that plant breeding or vegetable cell or tissue culture are used.

[0402.0.0.0] can be (Milner such as expression library (for example cDNA expression library), peptide, protein, nucleic acid, antibody, little organic compound, hormone, peptide mimics, PNA according to the inventive method test and compounds identified, Nature Medicine 1 (1995), 879-880; Hupp, Cell83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and the reference of quoting thereof).Described compound can also be the functional type derivative or the analogue of known inhibitor or activator.The method for preparing chemical derivative and analogue is well known to those skilled in the art, and be described in for example Beilstein, " Handbook of Organic Chemistry ", Springer editionNew York Inc., 175 Fifth Avenue, New York, N.Y.10010 U.S.A. and Organic Synthesis, Wiley, New York, USA.In addition, can test the effect of described derivative and analogue according to methods known in the art.In addition, peptide mimics and/or the computer aided design (CAD) of can (for example according to method mentioned above) using suitable derivative and analogue.The cell or tissue that uses in the inventive method is preferably host cell of the present invention, plant or the plant tissue of describing in the embodiment above.

[0403.0.0.0] therefore relates in another embodiment of the invention according to the method for identifying agonist of the present invention and obtaining or compounds identified, and described compound is the agonist of polypeptide of the present invention or polypeptide that the inventive method is used.

[0404.0.0.0] therefore, in one embodiment, the present invention relates to the method institute compounds identified that is tested and appraised The compounds of this invention in addition.

[0405.0.0.0] described compound is the homologue of polypeptide of the present invention for example.The homologue that can produce polypeptide of the present invention by the mutagenesis (as the point of discontinuity sudden change) or the brachymemma of polypeptide of the present invention.The variant form of term used herein " homologue " finger protein matter, described variant is taken on the effect of polypeptide active agonist of the present invention.Described proteinic agonist can keep essentially identical polypeptide biological activity of the present invention or its subclass.Particularly, described agonist is given expression of polypeptides level raising of the present invention and/or is expressed this agonist and give free and/or each fine chemicals raising of bonded in biology or its part in biological or its part.

[0406.0.0.0] the present invention relates to the antibody of specific recognition The compounds of this invention or agonist in one embodiment.

[0407.0.0.0] the invention still further relates to the diagnosis composition that contains at least one aforementioned nucleic acid molecule of the present invention, carrier, protein, antibody or compound and optional suitable means.

[0408.0.0.0] diagnosis composition of the present invention is applicable to that separating mRNA contacts the existence of the mRNA of resulting mRNA, detection and probe hybridization with the probe that usefulness under hybridization conditions contains the aforementioned nucleic acid probe from cell, and detects protein expression in the cell by this.The additive method that detects protein existence of the present invention comprises immunological technique known in the art, for example enzyme-linked immunosorbent assay.In addition, can also use nucleic acid molecule of the present invention as molecule marker in the plant breeding or primer.Suitable detection method is well known to those skilled in the art, for example the damping fluid of hybridization assays and solution (solution and damping fluid as the aforementioned) and be known as the method for traces such as Southern, the Western of descriptions such as Sambrook, Northern.

[0409.0.0.0] the present invention relates to contain nucleic acid molecule, carrier, host cell, polypeptide, antisense nucleic acid, antibody, vegetable cell, plant or plant tissue, can gather in the crops part, reproductive material and/or according to the test kit of the inventive method compounds identified or agonist or antagonist in another embodiment.

The compound of [0410.0.0.0] test kit of the present invention can (randomly with damping fluid and/or solution or in damping fluid and/or solution) be packaged in the container (as bottle).In the time of suitably, can in same container, pack one or more described components.In addition or alternatively, one or more described components can absorb in the solid support (as the hole of digest cellulose membrane filtration film, glass plate, chip or nylon membrane or microwell plate).This test kit can be used for any method described herein and embodiment, for example produce host cell, transgenic plant, pharmaceutical composition, detection homologous sequence, identify antagonist or agonist, as food or feed or as its additive, as the fill-in of plant treatment etc.

[0411.0.0.0] in addition, test kit can comprise the specification sheets that this test kit is used for the purposes of any described embodiment, has the purposes of biological or its part of the free of raising or each fine chemicals content of bonded especially for generation.

[0412.0.0.0] in one embodiment, described test kit contains coding one or more aforementioned proteinic other nucleic acid molecule and/or antibody, carrier, host cell, antisense nucleic acid, vegetable cell or plant tissue or plant.

[0413.0.0.0] in another embodiment, the present invention relates to produce the method for Pestcidal compositions, the step that it provides nucleic acid molecule of the present invention, carrier or polypeptide or is used for the polypeptide of the inventive method or comprises the inventive method of identifying described compound, agonist or antagonist, and with the form that can be used as the plant Pestcidal compositions make nucleic acid molecule of the present invention, carrier or polypeptide be used for the polypeptide of the inventive method or agonist identify according to the inventive method or process or use object compounds identified of the present invention.

[0414.0.0.0] in another embodiment, the present invention relates to produce the plant culturing method for compositions of supporting to produce " each fine chemicals ", it comprises the step of the inventive method and makes institute's compounds identified with the form that can be used as Pestcidal compositions.

[0415.0.0.0] " can be used as Pestcidal compositions " and is interpreted as the law of these compositions mycocide up to specification, plant nutrient, weedicide equal size.Preferred such composition does not have any infringement to shielded plant and the animal (comprising the mankind) that eats it.

The embodiment that [0416.0.0.0] the present invention is also relevant with relating to other purposes and method.Nucleic acid molecule as herein described, polypeptide, protein homologs, fusion rotein, primer, carrier, host cell can be used for one or more following methods: identify to can be used for plant and the associated biomolecule that each fine chemicals produces as described above; Genomic mapping; Identify and the location aim sequence; Study on Evolution; Determine the function desired zone; Regulation activity.

[0417.0.0.0] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of amino acid biosynthetic pathway is had resistance.Particularly, the expression excessively of polypeptide of the present invention may make plant resist and can block amino acid in the described plant (particularly each fine chemicals) synthetic weedicide.Inhibitor can suppress the one or more steps of methionine(Met) synthetic.Lys, Met and first committed step of Thr synthetic are first steps, i.e. aspartokinase enzyme catalysis aspartic acid phosphorylation forms the step of aspartoyl-b-phosphoric acid.Intestinal bacteria have 3 kinds of aspartokinase enzyme isoenzymes, and their correspondences aspect enzyme inhibition and feedback inhibition in 3 kinds of different aminoacids.Therefore the biosynthesizing of Methionin, methionine(Met) and Threonine is not as a group and Be Controlled.Approach from the aspartic acid to Methionin has 10 steps.Approach from the aspartic acid to the Threonine has 5 steps.Approach from the aspartic acid to the methionine(Met) has 7 steps.Article three, the regulation and control of approach also betide two branching-points:

-b-aspartic acid-semialdehyde (homoserine and Methionin)

-homoserine (Threonine and methionine(Met))

Regulation and control come from the feedback inhibition of the tapping point amino acid product shown in the top bracket.This organizes the important step of 3 amino acid in synthetic is the step that methionine synthases catalysis homocysteine is converted into methionine(Met):

In this reaction, homocysteine methyl formation methionine(Met), and the C1 donor is N5-methyl-THF.Therefore, the restraining effect (also comprising the supply of donor molecule) to one or more methionine synthases can suppress the synthetic of methionine(Met).

The example of amino acid synthetic weedicide is for example sulfonylurea and imidazolidinone weedicide in the blocking-up plant, the first step in these weedicide catalysis branched-chain amino acid biosynthesizing.Methionine(Met) synthetic inhibitor for example is described in Danishpajooh IO, 2001 Nitric oxide inhibitsmethionine synthase activity in vivo and disrupts carbon flow through thefolate pathway.J.Biol.Chem.276:27296-27303; Datko AH, 1982Methionine biosynthsis in Lemna-inhibitor studies.Plant Physiol.69:1070-1076; Lavrador K, 1998 A new series of cyclic amino acids asinhibitors of S-adenosyl L-methionine synthetase.Bioorg.Med.Chem.Lett.8：1629-1634；Thompson?GA，1982?Methionine?synthesis?in?Lemna-inhibition?of?cystathionine?gamma-synthase?by?propargylglycine.PlantPhysiol.70：1347-1352。In some biologies, the synthetic inhibition that is subjected to ethanol, lead, mercury, aluminium, Thiomersalate, copper, N2O of methionine(Met), for example as M.Waly, H.Oleteanu etc., 2004, Molecular Psychiatry discusses among the 1-13.

What is interesting is that when having DL-PGIY (a kind of special inhibitor of methionine(Met) synthetic), the mouseearcress seed germination is subjected to strong delay.And this kind compound suppresses growth of seedling fully.In case in the germination substratum, add methionine(Met) then greatly alleviate these phenotypic effects.These results show, methionine synthases and S-adenosylmethionine synthetic enzyme are the metabolic basal component of control the transition from resting stage to the high activity state during the seed germination.And, in the temporary transient pattern of viewed these protein accumulations and the mouseearcress only after radicle stretches out the basic role of endogenous ethene be consistent; See Gallarado, K., 2002, Importance ofmethionine biosynthesis for Arabidopsis seed germination and seedlinggrowth, Physiolgia Plantarum, 116 (2), pp 238-247.Therefore, polypeptide of the present invention cross expressing in plant can protective plant be avoided weedicide to suppress methionine(Met) synthetic.

[0418.0.0.0] therefore, nucleic acid molecule of the present invention serves many purposes.At first, they can be used for identification of organism or its close relative.In addition, they also can be used for its existence or its close relative in Identifying micro-organisms or the plant mixed population.By under stringent condition, detecting the genomic dna that from the culture of single or mixed plant population, extracts, can determine whether to have used the present invention or not have biological or its close relative with the probe of crossing over the exclusive zone of gene of the present invention.

[0419.0.0.0] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor can be with the sequence of affinity species abundant homology, thereby these nucleic acid molecule can serve as a mark in making up the mapping of relationship biological gene group.

[0420.0.0.0] therefore the present invention relates to producing the method that each fine chemicals cultivates plants, and it comprises:

(a) provide the first kind of plant mutation of that produce according to the inventive method, preferred (mistakes) expression nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor;

(b) with the first kind of plant mutation and the second kind of plant intervarieted cross; With

(c) be marked at the progeny plant that ability that distribution in the filial generation of representing the first kind of plant mutation and (excessive) thereof produce fine chemicals is selected excessive each fine chemicals of generation by analyzing molecules.

About the details of the purposes of molecule marker in breeding be found in Kumar etc. 1999 (BiotechAdv., 17:143-182) and Peleman and van der Voort 2003 (Trends Plant Sci.2003 Jul; 8 (7): 330-334).Molecule marker can for example relate to nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor and/or its expression level.Therefore, molecule marker can be to be used to identify that the genome of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor exists or localized probe of genome or PCR primer sets, for example in Southern engram analysis or PCR or its expression level (being Northern engram analysis or quantitative PCR).Therefore, in one embodiment, the nucleic acid molecule that the present invention relates to nucleic acid molecule of the present invention or code book invention polypeptide is used for the purposes of breeding as molecule marker, especially for cultivating the species that each high or low fine chemicals is produced.

[0421.0.0.0] nucleic acid molecule of the present invention can also be used for evolving and protein structure research.Can assess interbiotic evolution sibship by relatively the present invention or the employed sequence of the inventive method and sequence from the similar enzyme of coding of other biological.Similarly, guard in which zone of such comparison in can assessment sequence, and which then is not that this can help to determine the necessary protein of the function zone of enzyme.This type determine that research is valuable to protein engineering, and may provide about protein and can bear which kind of mutagenesis and the prompting of not loss of function.

[0422.0.0.0] therefore, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor can be used to identify gives other nucleic acid molecule that each fine chemicals increases after expression.

[0423.0.0.0] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or the gene fragment of giving polypeptide of the present invention or being used for the expression of polypeptides (preferably containing nucleic acid molecule of the present invention) of the inventive method can be used for the derive integrating map of proterties of marker-assisted breeding or each fine chemicals.

[0424.0.0.0] therefore, nucleic acid of the present invention, polypeptide of the present invention or be used for the polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, agonist with the inventive method evaluation, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce fine chemicals and one or more other amino acid, particularly Threonine, L-Ala, glutamine, L-glutamic acid, Xie Ansuan, l-asparagine, phenylalanine, leucine, proline(Pro), tryptophane, tyrosine, Xie Ansuan, Isoleucine and arginine.Therefore, nucleic acid of the present invention or nucleic acid molecule or its complementary sequence, the polypeptide of the present invention identified with the inventive method or be used for the polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, each fine chemicals that antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing biological or its part (as cell).

[0425.0.0.0] in addition, nucleic acid of the present invention, polypeptide of the present invention or be used for the polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the antagonist identified with the inventive method or agonist, antibody of the present invention, antisense molecule of the present invention or the nucleic acid molecule identified with the inventive method can be used to prepare Pestcidal compositions.

[0426.0.0.0] in addition, nucleic acid of the present invention, polypeptide of the present invention or be used for the polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist or agonist with the inventive method evaluation, antibody of the present invention, antisense molecule of the present invention or can be used for identifying and produce the compound that to give biological or each fine chemicals level of its part of regulation and control with the nucleic acid molecule that the inventive method is identified, if described compounds identified is applied to biology or its part (promptly as the part of its food or in cultivation or substratum), preferably identifies and produce and give the compound that each fine chemicals level improves in biology or its part.

[0427.0.0.0] these and other embodiment is disclosed, and is included among specification sheets of the present invention and the embodiment.Can use as electronics and from the public library, retrieve about treating other documents of any used according to the present invention method, purposes and compound.For example can utilize the public database " Medline " that provides on the internet, for example http://www.ncbi.nlm.nih.gov/PubMed/medline.html.Other databases and address such as http://www.ncbi.nlm.nih.gov/, http://www.infobiogen.fr/, http://www.fmi.ch/biology/research-tools.html, http://www.tigr.org/ are for it be known to those skilled in the art that and can using for example http://www.lycos.com acquisition.Berks, TIBTECH 12 (1994), provided the general introduction of the biotechnology patent information that is used for RS and current awareness and the investigation of patent information related resource among the 352-364.

[0428.0.0.0] table 1 provides the overview about disclosed sequence among the present invention.

1) raising of metabolite:

Max: maximum x-times (wild-type is carried out normalization method)-

Min: minimum x-is (carrying out normalization method to wild-type) doubly

2) reduction of metabolite:

Max: maximum x-is (wild-type is carried out normalization method) (minimum reduction) doubly

Min: minimum x-is (wild-type is carried out normalization method) (maximum reduction) doubly

[0429.0.0.0] the present invention describes by following examples.Present embodiment is for example understood the basic invention that is intended to not limit object of the present invention.The full content of the reference of quoting in the present patent application, patent application, patent and publication patent application is incorporated herein by reference in this article.

[0430.0.0.0] embodiment

[0431.0.0.0] embodiment 1: in intestinal bacteria, clone

[0432.0.0.0] uses the method for known good foundation (to consult as Sambrook, J. etc. (1989) " Molecular Cloning:A Laboratory Manual " Cold Spring HarborLaboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Mole-cular Biology ", John Wiley ﹠amp; Sons), the DNA polynucleotide that will have shown in Table I the 5th row sequence and coding as a listed polypeptide of following table 1 be cloned into plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl Acad.Sci.USA, 75:3737-3741); PACYC 177 (Change ﹠amp; Cohen (1978) J.Bacteriol.134:1141-1156); PBS series plasmid (pBSSK+, pBSSK-and other; Stratagene, LaJolla, USA) or clay such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J.Rosenthal, A., and Waterson, R.H. (1987) Gene 53:283-286) with at expression in escherichia coli.

[0433.0.0.0] embodiment 2:DNA order-checking and computer function analysis

[0434.0.0.0] is by standard method, particularly use the chain mensuration method of ABI377 sequenator that dna sequencing (is consulted as Fleischman, (1995) Whole-genome RandomSequencing and Assembly of Haemophilus Influenzae Rd. such as R.D., Science 269; 496-512).

[0435.0.0.0] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.0.0] can be by changing plasmid DNA (or other carrier DNAs) over to the intestinal bacteria that can not keep its genetic information integrity or other microorganisms (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of Corynebacterium glutamicum, to produce each fine chemicals.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms inEscherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that [0436.1.0.0] external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.0.0] embodiment 4: the DNA between intestinal bacteria and Corynebacterium glutamicum shifts

[0438.0.0.0] some excellent bacillus and tyrothricin species contain the endogenous plasmid (as pHM1519 or pBL1) of self-replicating (consulting as Martin the summary of J.F. etc. (1987) Biotechnology 5:137-146).Can use for the shuttle vectors of intestinal bacteria and Corynebacterium glutamicum and to have added the replication orgin that is used for Corynebacterium glutamicum or from the standard escherichia coli vector (Sambrook of the suitable mark of Corynebacterium glutamicum, J. etc., (1989), " Molecular Cloning:A LaboratoryManual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from isolating endogenous plasmid from excellent bacillus and tyrothricin species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " FromGenes to Clones-Introduction to Gene Technology ", VCH, Weinheim) gene.There is the multiple example that in intestinal bacteria and Corynebacterium glutamicum, duplicates and can be used for the shuttle vectors of multiple purpose (comprising expressing gene) of preparation (to consult in the document as Yoshihama, (1985) J.Bacteriol.162:591-597 such as M., Martin, J.F. etc., (1987) Biotechnology, 5:137-146 and Eikmanns, B.J. etc. (1992) Gene 102:93-98).The suitable carrier that duplicates in excellent bacillus is for example pZ1 (Menkel etc., Appl.Environ.Microbiol., 64,1989:549-554), pEkEx1 (Eikmanns etc., Gene 102,1991:93-98) or pHS2-1 (Sonnen etc., Gene 107,1991:69-74).These carriers are based on cryptic plasmid pHM1519, pBL1 or pGA1.Can also use other plasmid vectors by the same manner, as based on pCG4 (US4,489,160), pNG2 (Serwold-Davis etc., FEMS Microbiol.Lett., 66,1990:119-124) or the plasmid of pAG1 (US 5,158,891).

[0439.0.0.0] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the Corynebacterium glutamicum strain of aforementioned shuttle vectors.Can pass through protoplast transformation (Kastsumata, R. etc., (1984) J.Bacteriol.159,306-311), electroporation (Liebl, E. etc., (1989) FEMS Microbiol.Letters, 53:399-303) and use under the situation of specific support joint (for example

A., wait described in (1990) J.Bacteriol.172:1663-1666) realize the Corynebacterium glutamicum conversion.Similarly, can transform into intestinal bacteria and the shuttle vectors of Corynebacterium glutamicum is transferred to intestinal bacteria from the plasmid DNA of Corynebacterium glutamicum and with it by (using standard technique known in the art) preparation.Can use standard method to implement this step of converting, but preferably use Mcr-defective escherichia coli bacterial strain, as NM522 (Gough ﹠amp; Murray (1983) J.Mol.Biol.166:1-19).

Also there is standard technique known to the skilled if the sequence that transforms need advantageously be integrated into excellent bacillus gene group in [0440.0.0.0] for this purpose.The example that is used for this purpose be used to duplicate and the hom-thrB operon that increases as (Appl.Environ.Microbiol., 60,1994:126-132) described plasmid vectors such as Remscheid.In the method, complete genome is cloned in the plasmid vector that can in host (as intestinal bacteria), duplicate but in Corynebacterium glutamicum, not duplicate.Suitable carriers for for example pSUP301 (Simon etc., Bio/Technology 1,1983:784-791), pKIBmob or pK19mob (

Deng, Gene 145,1994:69-73), pGEM-T (Promega Corp., Madison, WI, USA), pCR2.1-TOPO (Schuman, J.Biol.Chem., 269,1994:32678-32684, US 5,487,993),

(Invitrogen, Groningen, Holland) or pEM1 (Schrumpf etc., J.Bacteriol., 173,1991:4510-4516).

[0441.0.0.0] embodiment 5: measure the expression of mutant/transgenic protein

Suddenly change in [0442.0.0.0] observation transformed host cell or transgenic protein activity reaches this fact in the mode similar to wild-type protein with similar scale based on protein.Measuring appropriate method that mutant gene or transgenosis transcribe quantity (can for the sign of the mRNA quantity of translation gene product) (consults as Ausubel etc. for carrying out the Northern trace, (1988) " Current Protocols inMolecular Biology ", Wiley:New York), wherein provide primer to design with goal gene bonded mode with detectable mark (being generally radioactivity or chemiluminescent labeling), thereby as the total RNA that extracts the biological culture thing, in glue, separate, be applied to stable matrix and when hatching, the combination of probe and indicate the existence and the quantity of this gene mRNA in conjunction with quantity with this probe.Other method is a quantitative PCR.This information detects the degree of genetic transcription.Can from Corynebacterium glutamicum or other microorganism, separate total cell RNA with several different methods known in the art (as Bormann, E.R. etc. are described in (1992) Mol.Microbiol.6:317-326).

[0443.0.0.0] can use standard technique such as Western trace to measure from the proteinic existence or the relative quantity of this mRNA translation and (consult as (1988) such as Ausubel " Current Protocols inMolecular Biology ", Wiley, New York).In this method, extract total cell protein matter, separate by gel electrophoresis, transfer to matrix such as nitrocellulose and with probe (as with target protein matter specificity bonded antibody) hatch.This probe directly or indirectly provides with the chemoluminescence or the colorimetric mark of easy detection usually.The existence and the quantity of purpose mutein in the existence of mark and the observed schedule of quantities clear-cells.Yet additive method also is known.

[0444.0.0.0] embodiment 6: the Corynebacterium glutamicum of cultivating genetic modification: substratum and culture condition

[0445.0.0.0] cultivates the excellent bacillus of genetic modification in synthetic or natural medium.Multiple different substratum for excellent bacillus are known, and can extensively obtain (Lieb etc. (1989) Appl.Microbiol.Biotechnol.32:205-210; (1998) BiotechnologyLetters 11:11-16 such as von der Osten; Patent DE 4 120 867; Liebl (1992) " The Procaryotes ", volume II Balows, A. waits volume, " the The GenusCorynebacterium " among the Springer-Verlag).

[0446.0.0.0] can be used according to the invention described substratum form by one or more carbon sources, nitrogenous source, inorganic salt, VITAMIN and trace element usually.Preferred carbon source is a sugar, as monose, disaccharide or polysaccharide.Extraordinary carbon source is glucose, fructose, seminose, semi-lactosi, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or Mierocrystalline cellulose.Can also sugar be added in the substratum by the byproduct that complex chemical compound (as molasses) or other asccharin are practiced.The mixture that can also advantageously add several kinds of carbon source.Other possible carbon sources are alcohol and/or organic acid (as methyl alcohol, ethanol, acetate or lactic acid).Nitrogenous source is generally the organic or inorganic nitrogen compound or contains the material of described compound.The example of nitrogenous source comprises ammonia, ammoniacal liquor or ammonium salt such as NH ₄Cl or (NH ₄) ₂SO ₄, NH ₄OH, nitrate, urea, amino acid or compound nitrogen source (as corn steep liquor, soyflour, soybean protein, yeast extract, meat extract etc.).Can advantageously use the mixture of aforementioned nitrogenous source.

The inorganic salt compound that can contain in [0447.0.0.0] substratum comprises muriate, phosphoric acid salt or the vitriol of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Can in substratum, add sequestrant to preserve the solution metal ion.Suitable sequestrant specifically comprises dihydroxy phenol (as pyrocatechol or former benzoic acid) or organic acid (as citric acid).Substratum also contains other somatomedins such as VITAMIN or growth stimulant (comprising biological example element, riboflavin, thiamines, folic acid, nicotinic acid, pantothenate (panthothenate) and pyridoxol) usually.Somatomedin and salt are often from complex medium component (as yeast extract, molasses, corn steep liquor etc.).The accurate composition of the compound that uses in the substratum depends on concrete experiment to a great extent, and each particular case is determined separately.The information of optimizing substratum be found in textbook " Applied Microbiol.Physiology, A PracticalApproach " (P.M.Rhodes, P.F.Stanbury compiles, IRL Press (1997) S.53-73, ISBN 0 19 963,577 3).Can also obtain growth medium from supplier, for example Standard 1 (Merck) or BHI (brain heart leach liquor, DIFCO) etc.

[0448.0.0.0] all nutrient media componentses are sterilized by heating (1.5bar, 121 ℃ following 20 minutes) or filtration sterilization.Component can be sterilized or sterilization respectively in case of necessity together.All nutrient media componentses can exist when cultivation is initial or continue or adding in batches by required.

[0449.0.0.0] defines culture condition respectively to each experiment.Temperature generally also can keep constant or can change in experimentation between 15 ℃ and 45 ℃.The pH of substratum should be in 5 to 8.5 scope (preferred about 7.0), and can keep by add damping fluid in substratum.Example for the damping fluid of this purpose is a potassium phosphate buffer.Synthetic damping fluid (as MOPS, HEPES, ACES etc.) can be used as and replaces using or using simultaneously.Can be by adding as NaOH or NH ₄OH keeps culture pH value constant in incubation period.If use complex medium component (as yeast extract), the extra damping fluid of then less needs is because many complex chemical compounds have high surge capability.When using the fermentor cultivation microorganism, can also use gaseous ammonia to regulate pH.

[0450.0.0.0] incubation period, arrive in several days the scope in a few hours usually.Interval when selecting this in the mode of accumulation maximum product in the fermented liquid.Disclosed growth experiment can carry out in a plurality of containers, for example the glass of microtiter plate, Glass tubing, vial or various size or metal fermentor tank.For screening a large amount of clones, should or shake culturing micro-organisms in the bottle (simple bottle or baffle plate bottle) at microtiter plate, Glass tubing.The preferred 100ml of the required growth medium of 10% (based on volume) of packing into that uses shakes bottle.Should go up at orbital shaker (amplitude 25mm) and rock bottle with the speed in the scope from 100 rev/mins to 300 rev/mins.Can reduce vaporization losses by keeping damp atmosphere, perhaps can carry out the mathematics correction vaporization losses.

[0451.0.0.0] if detect the clone of genetic modification, also should comprise the contrast clone that contains the basic plasmid that has or not insertion or the contrast clone of unmodified in the test.If the express transgenic sequence also should advantageously comprise the contrast clone in the test.Use is at 30 ℃ of agar plates of hatching, as cultured cells on the CM flat board (the poly-peptone of 10g/l glucose, 2.5g/l NaCl, 2g/l urea, 10g/l, 5g/l yeast extract, 5g/l meat extract, 22g/l agar transfer to pH value 6.8 with 2M NaOH) advantageously substratum is hatched to OD600 be 0.5 to 1.5.For example come inoculation medium by importing pre-incubated seed biology.

[0451.1.0.0] for example comes inoculation medium by importing from the salts solution of the Corynebacterium glutamicum cell of CM flat board or by the pre-culture of the liquid that adds this bacterium.

[0452.0.0.0] embodiment 7: the coded proteinic function of analyzed in vitro transforming sequence

Being determined as of activity of [0453.0.0.0] enzyme and kinetic parameter is known in the art.The experiment of measuring specific modification enzyme activity must be fit to the specific activity of wild-type, and this is that the technician can accomplish fully.The introduction of enzyme and be found in for example following document: Dixon, M. and Webb, E.C:(1979) " Enzymes ", Longmans, London about the general introduction of the detail of structure, kinetics, principle, method, application and the active example of evaluation plurality of enzymes; Fersht (1985) " EnzymeStructure and Mechanism ", Freeman, New York; Walsh (1979) " Enzymatic Reaction Mechanisms ", Freeman, San Francisco; Price, N.C., Stevens, L. (1982) " Fundamentals of Enzymology ", Oxford Univ.Press:Oxford; Boyer, P.D compiles (1983) The Enzymes, third edition .AcademicPress, New York; Bisswanger, H. (1994) Enzymkinetik, second edition VCH, Weinheim (ISBN 3527300325); Bergmeyer, H.U., Bergmeyer, J., Gra β l, M. compiles (1983-1986) " Methods of Enzymatic Analysis ", the third edition, I-XII volume, Verlag Chemie:Weinheim; " Ullmann ' s Encyclopedia of IndustrialChemistry " (1987) A9 volume, " Enzymes ", VCH, Weinheim, 352-363 page or leaf.

[0454.0.0.0] embodiment 8: the influence that analyzing nucleic acid molecules produces amino acid

[0455.0.0.0] can be by cultivating down the microorganism of modifying and analyzing substratum and/or the influence that genetic modification produces amino acid in the Corynebacterium glutamicum is measured in raising that the amino acid of cellular component produces at felicity condition (as indicated above).These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

[0456.0.0.0] also can analyze other components (as intermediate product and by product) of the pathways metabolism that is used for producing described compound except measuring the fermentation end product, with the yield of measuring biological total output, compound and/or produce efficient.Analytical procedure comprises amount (for example sugar, carbohydrate, nitrogenous source, phosphoric acid salt and other ions), the mensuration biomass composition of measuring nutrient substance in the substratum and grows, analyzes the generation of biosynthetic pathway eubolism thing and measure the gas that produces between yeast phase.These standard methods are described in " Applied Microbial Physiology; A PracticalApproach ", P.M.Rhodes and P.F.Stanbury compile IRL Press, 103-129 page or leaf; 131-163 page or leaf and 165-192 page or leaf (ISBN:0199635773) and the reference of quoting thereof.

[0457.0.0.0] embodiment 9: amino acid purifying

[0458.0.0.0] can reclaim amino acid from cell or from the supernatant liquor of aforementioned culture by multiple methods known in the art.For example, at first reclaim culture supernatants.For this reason, by at a slow speed centrifugal from culture harvested cell.General by standard technique (as mechanical force or supersound process) destruction or lysing cell.By centrifugal removal cell debris, supernatant liquor part (suitably time with culture supernatants) is used to be further purified amino acid.Yet, if amino acid exists with sufficiently high concentration in supernatant liquor, also can the individual curing supernatant liquor.In this case, can be for example by extracting and/or salt precipitation or be further purified amino acid or aminoacid mixture by ion-exchange chromatography.

[0459.0.0.0] if necessary and need can continue other chromatographic steps with suitable resin, and wherein amino acid in the sample rather than pollutent are stayed on the chromatography resin, and perhaps pollutent rather than the sample that contains product (amino acid) are stayed on the resin.In case of necessity, can use same or other chromatography resins repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and at the most efficient use of specific molecular to be purified.Can be by filtering or the product of ultrafiltration and concentration purifying and be stored in the temperature that guarantees the product maximum stable.The multiple purification process that is not limited to above purification process is known in this field.They are described in for example Bailey, J.E.﹠amp; Ollis, D.F.BiochemicalEngineering Fundamentals, McGraw-Hill:New York (1986).

[0460.0.0.0] can pass through isolating amino acid whose identity of this area measured by standard techniques and purity.They comprise high performance liquid chromatography (HPLC), spectrography, mass spectrum (MS), dyeing process, thin-layer chromatography, NIRS, enzyme assay or microbiological analysis.These analytical procedures are compiled in (1994) Appl.Environ.Microbiol.60:133-140 such as Patek; Malakhova etc. (1996) Biotekhnologiya 11:27-32; With (1998) Bioprocess Engineer.19:67-70.Ulmann ' s Encyclopedia of Industrial Chemistry such as Schmidt " (1996) A27 volume, VCH:Weinheim, 89-90 page or leaf, 521-540 page or leaf, 540-547 page or leaf, 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlasof Biochemistry and Molecular Biology ", John Wiley and Sons; Fallon, A. etc. (1987) " Laboratory Techniques in Biochemistry and MolecularBiology ", " the Applications of HPLC in Biochemistry " in 17 volumes.

[0461.0.0.0] embodiment 10: clone SEQ ID NO:1 is used for expressing plant

[0462.0.0.0] uses people such as Sambrook, Molecular Cloning:Alaboratory manual, Cold Spring Harbor 1989, the standard method of describing among the Cold Spring HarborLaboratory Press unless stated otherwise.

[0463.0.0.0] passes through pcr amplification SEQ ID NO:1 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

The composition of [0464.0.0.0] Pfu Turbo archaeal dna polymerase schedule of operation is as follows: 1x PCR damping fluid (Stratagene), every kind of dNTP of 0.2mM, 100ng yeast saccharomyces cerevisiae (bacterial strain S288C; ResearchGenetics, Inc. now is Invitrogen) or intestinal bacteria (bacterial strain MG1655; E.coli GeneticStock Center) genomic dna, 50pmol forward primer, 50pmol reverse primer, 2.5uPfu Turbo archaeal dna polymerase.Amplification cycles is as follows:

[0465.0.0.0] 94-95 ℃ of circulation in 3 minutes, 95 ℃ of 1 minute or 94 ℃ 30 seconds, 50 ℃ 45 seconds, 50 ℃ 30 seconds or 55 ℃ 30 seconds and 72 ℃ of 210-480 seconds then, 28-36 circulation under each situation, 72 ℃ of circulations in 8 minutes then, 4 ℃ then.The composition of Herculase polysaccharase schedule of operation is as follows: 1x PCR damping fluid (Stratagene), every kind of dNTP of 0.2mM, 100ng yeast saccharomyces cerevisiae (bacterial strain S288C; Research Genetics, Inc. now is Invitrogen) or intestinal bacteria (bacterial strain MG1655; E.coli Genetic Stock Center) genomic dna, 50pmol forward primer, 50pmol reverse primer, 2.5u Herculase polysaccharase.Amplification cycles is as follows:

[0466.0.0.0] 94 ℃ of 2-3 minute circulation, ℃ 30 seconds and 72 ℃ 10 minutes 94 ℃ of 30 seconds, 55-60 then, 25-30 circulation under each situation, 72 ℃ of circulations in 10 minutes then, 4 ℃ then.

[0467.0.0.0] selects following primer sequence for genes of SEQ ID NO:1:

I) forward primer (SEQ ID NO:3):

ATGGAACAGAACAGGTTCAAGAAAG

Ii) reverse primer (SEQ ID NO:4):

TTACAGTTTTTGTTTAGTCGTTTTAAC

[0468.0.0.0] after this uses the QIAquick post according to Standard operation procedure SOP (Qiagen) purifying amplified production.

During PCR product that [0469.0.0.0] clone produces via Pfu Turbo archaeal dna polymerase, according to Standard operation procedure SOP with SmaI (MBI Fermentas) restricted cut vector DNA (30ng) and add the high-salt buffer termination.The carrier segments of restricted cutting is used Standard operation procedure SOP (Macherey-Nagel) purifying by the Nucleobond post.After this, linearizing carrier is according to Standard operation procedure SOP (MBI Fermentas) dephosphorylation.

[0470.0.0.0] directly will enter in the treated binary vector by the PCR product cloning that Pfu Turbo archaeal dna polymerase produces.The PCR product that utilizes the T4 archaeal dna polymerase and use Standard operation procedure SOP (for example MBI Fermentas) phosphorylation to produce by Pfu Turbo archaeal dna polymerase, and the clone enters in the binary vector of treated mistake.

[0471.0.0.0] uses Pfu Turbo archaeal dna polymerase benefit flat in the second step building-up reactions by the DNA end of the PCR product that the Herculase archaeal dna polymerase produces.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Then in the carrier that also the PCR product cloning is advanced to handle.Utilize Pfu Turbo archaeal dna polymerase, in second building-up reactions, mend the flat DNA end that passes through the PCR product of HerculaseDNA polysaccharase generation.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Utilize the T4 archaeal dna polymerase and use Standard operation procedure SOP (for example MBI Fermentas) phosphorylation PCR product, and be cloned in the carrier of handling.

[0472.0.0.0] uses the binary vector that comprises the terminator sequence between expression cassette, clone's box and the T-DNA border sequence of selecting box (promotor, selective marker, terminator) and having promotor.Except that clone's those in box, binary vector does not contain the SmaI cleavage site.Spendable binary vector is that those skilled in the art are known; The general introduction of binary vector and uses thereof is found in Hellens, R., Mullineaux, P. and Klee H., [(2000), A guide to Agrobacterium binaryvectors, Trends in Plant Science, 5 (10): 446-451.According to employed carrier, also can advantageously finish the clone by other Restriction Enzymes.Can suitable favourable cleavage site be added among the ORF by using suitable pcr amplification primer.

[0473.0.0.0] mixes the prepared amplified production of prepared carrier of about 30ng and set amount and is connected by adding ligase enzyme.

[0474.0.0.0] adds competence Bacillus coli cells (bacterial strain DH5 α) and hatched 20 minutes at 1 ℃ in same reaction vessel, then 42 ℃ of heat shocks 90 seconds and be cooled to 4 ℃, institute's connection carrier is transformed.Add perfect medium (SOC) then and mixture was hatched 45 minutes at 37 ℃.Then whole mixtures are coated on the agar plate that contains microbiotic (selecting) and in 37 ℃ of overnight incubation according to use binary vector function.

The result of [0475.0.0.0] clone step is by the amplification checking, and the primer that is combined in the integration site upstream and downstream is used in amplification, thereby allows the amplification inset.In addition, in the PCR reaction, use the combination of said gene special primer and upstream and downstream primer, the clone who has correct direction of insertion with evaluation.As described in Taq archaeal dna polymerase (Gibco-BRL) schedule of operation, increase.

[0476.0.0.0] amplification cycles is as follows: 94 ℃ of circulations in 5 minutes, 94 ℃ of 15 seconds, 55-60 ℃ 15 seconds and 72 ℃ are 5 minutes then, 35 circulations under each situation, 72 ℃ of circulations in 10 minutes then, 4 ℃ then.

[0477.0.0.0] checked several clones, but has only the clone who detects the big or small PCR product of expectation therein to be used for following step.

The part of [0478.0.0.0] this positive colony shifts in the reaction vessel of adding perfect medium (LB) into and 37 ℃ of overnight incubation.This LB substratum contain selection be suitable for the binary vector that uses (seeing above) and the microbiotic of existing resistant gene wherein, to select the clone.

[0479.0.0.0] is as preparation plasmid as described in the Qiaprep Standard operation procedure SOP (Qiagen).

[0480.0.0.0] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:1

[0481.0.0.0] transforms the isolating plasmid DNA of 1ng in the agrobacterium tumefaciens bacterial strain GV 3101 pMP90 competent cells (Koncz and Schell, Mol.Gen.Gent.204,383-396,1986) by electroporation.The selection of agrobacterium strains depends on the selection of binary vector.The possible bacterial strain and the general introduction of feature thereof are found in Hellens, R., Mullineaux, P. and Klee H., (2000), Aguide to Agrobacterium binary vectors, Trends in Plant Science, 5 (10): 446-451.After this, adding perfect medium (YEP) and mixture shifted into hatched 3 hours at 28 ℃ in the new reaction container.After this, all reaction mixtures are coated on the YEP agar plate that has added microbiotic separately (for example to GV3101 pMP90 with Rifampin and gentamicin), and another microbiotic of selecting based on binary vector of coating, and hatched 48 hours at 28 ℃.

[0482.0.0.0] uses the Agrobacterium that comprises the plasmid construction body that produces among the embodiment 10 to be used for Plant Transformation then.

[0483.0.0.0] chooses bacterium colony and joins the 3ml liquid TB substratum from agar plate by pipette tip, and this substratum still comprises the suitable microbiotic that depends on agrobacterium strains and binary plasmid.Pre-culture was cultivated 48 hours down at 28 ℃ and 120 rev/mins.

[0484.0.0.0] 400ml contains antibiotic LB substratum same as above and is used for main culture.Then pre-culture is shifted into main culture, grew 18 hours down at 28 ℃ and 120 rev/mins.4000 rev/mins resuspended in infiltration substratum (MS substratum, 10% sucrose) with throw out after centrifugal.

[0485.0.0.0] is used for plant transformed in order to cultivate, with plate (Piki Saat 80, green, provide with filter screen bottom, 30x 20x 4.5cm is from Wiesauplast, Kunststofftechnik, Germany) partly be full of with GS90 matrix (standard soil, Werkverband E.V., Germany).Proplant solution with 0.05% (Chimac-Apriphar, Belgium) spends the night to plate and adds water.Seed (Nottingham Arabidopsis Stock Centre, UK with Arabidopis thaliana C24; NASC Stock N906) is dispersed on the plate, approximately 1000 seeds of each plate.With plate on the cover cap and place stratification equipment (8h, 110 μ μ mol/m ²/ s ^-1, 22 ℃; 16h, dark, 6 ℃) in.After five days, plate is placed short day phytotron (8h 130 μ mol/m ²/ s ^-1, 22 ℃; 16h, dark, 20 ℃), they keep forming up to rough leaf in about 10 days there.

[0486.0.0.0] seedling is transferred in the basin that comprises same matrix (Teku pots, 7cm, LC series, manufacturers is

GmbH ﹠amp; Co, Germany).Every basin moves into five plants.Then basin is put back to the short day phytotron and made the plant continued growth.

After [0487.0.0.0] 10 days, plant is shifted into (supplementary illumination, 16h, 340 μ E, 22 ℃ in the greenhouse; 8h, dark, 20 ℃), regrowth 17 days.

[0488.0.0.0] floods the big arabidopsis thaliana of six weeks that just begins to bloom 10 seconds in the agrobacterium suspension that above-mentioned previous usefulness 10 μ l Silwett L77 (Crompton S.A., Osi Specialties, Switzerland) handled in order to transform.Described method is described in Clough and Bent, 1998 (Clough, JCand Bent, AF.1998Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant is J.16:735-743.

[0489.0.0.0] places plant 18 hours in moisture chamber subsequently.After this, basin is sent back to the greenhouse and make the plant continued growth.Plant kept for 10 weeks up to gathering in the crops seed in the greenhouse again.

According to being used to select to transform the resistance marker that plant is used, the seed of results is cultivated in the greenhouse and is carried out spray and select [0490.0.0.0], perhaps sterilizes earlier to plant then and is adding separately on the agar plate of selective reagents.With

Resistance is an example, with 2 to 3 days intervals with 0.02%

With seedling spraying four times, and allow transgenic plant bear seed.The seed of transgenic arabidopsis plant is kept at (20 ℃) in the refrigerator.

[0491.0.0.0] embodiment 12: be used for the plant growing of bioanalysis

[0492.0.0.0] for the bioanalysis of transgenic plant, the latter is growth equably in specific cultivating device.For this reason, GS-90 matrix is inserted in the dress basin machine (LaibleSystem GmbH, Singen, Germany) and in the basin of packing into as compost mixture.After this, 35 basins are combined in the dish also with Pu Like (Previcur) processing.During processing, general the making every effort to overcome of 25ml injected the 10l tap water.Such quantity is enough handled about 200 basins.Basin is placed Pu Like solution, and use the tap water that does not contain Pu Like additionally to irrigate top.They used within four days.

During [0493.0.0.0] sowing, the seed that will once be stored in the refrigerator (20 ℃) shifts out and shifts in the basin that compost into is housed from the Eppendorf pipe with toothpick.All about 5-12 seed thrown in the into middle part of basin.

[0494.0.0.0] after planting, the plate that will have a basin cover with the plastic cover of coupling and in the stratification chamber in 4 ℃ of dark placements 4 days.Humidity is about 90%.After the stratification, experimental plant with dark week of illumination in 16 hours, 8 hours aspire to 20 ℃, 60% atmospheric moisture and the about CO of 400ppm ₂Growth is 22-23 days under the concentration.The light source that uses be the Powerstar HQI-T 250W/D fluorescent lamp from Osram, and the about 220 μ E/m2/s-1 of its generation light intensity are similar to sun stratographic light.

[0495.0.0.0] carried out resistance marker with it and selects when plant-growth to 8,9 and 10 day age.0.015% (volume/volume) with 1l

(Glufosinate-ammonium) aqueous solution (Aventis Cropsience, Germany) is handled about 1400 basin transgenic plant.After 3-4 days, can know and distinguish transgenosis resistance seedling (seedlings of 4 leaf phases) and unconverted seedling.Albefaction of non-transgenic seedling or death.When the transgenosis resistance plant arrives 14 day age, with they thinnings.The plant best at the basin interstitial growth is considered to target plant.All remaining plants use the metal tweezers carefully to remove and abandon.

[0496.0.0.0] during plant-growth, use distilled water is irrigated (watering to compost) and is watered to placing and carry out the bottom irrigation in the groove on top.When reaching 23 day age, plant gathers in the crops.

[0497.0.0.0] embodiment 13: the metabolic analysis of transgenic plant

The modification to above-mentioned metabolite content that [0498.0.0.0] identifies according to the present invention is identified according to following step.

A) sampling of sample and preservation

[0499.0.0.0] sampling is directly carried out in the controlled environment chamber.Use little laboratory scissor cut plant,, move in the extraction tube of precooling and place with on the aluminium frame of cooled with liquid nitrogen with laboratory balance quick weighing.If desired, extraction tube can be kept in-80 ℃ of refrigerators.Amount to not more than 10 to 20 seconds from shearing plant to the time that consumes between being chilled in it liquid nitrogen.

B) freeze-drying

[0500.0.0.0] notes plant is kept at deep refrigeration state (temperature＜-40 ℃) or lyophilize up to the solvent contact first time in experiment.

The aluminium frame that [0501.0.0.0] will hold the extraction tube of plant sample is placed in the freeze-drier of precooling (40 ℃).Starting temperature in the main drying stage is 0.120mbar for-35 ℃ and pressure.At drying stage, parameter is according to the pressure and temperature change of program.Outlet temperature after 12 hours is 0.001-0.004mbar for+30 ℃ and resulting pressure.After closing vacuum pump and refrigerator, system is washed with air (by the drying tube drying) or argon gas.

C) extract

After lyophil(e)apparatus was rinsed, the 5ml that the extraction tube that the lyophilized vegetable material will be housed at once is transferred to ASE equipment extracted in the tube (the accelerated solvent extractor ASE 200 (DIONEX) that has solvent controller and AutoASE software) [0502.0.0.0].

24 sample positions of [0503.0.0.0] ASE equipment (the accelerated solvent extractor ASE 200 (DIONEX) that has solvent controller and AutoASE software) are placed plant sample, comprise that some are used for the sample of test mass control.

[0504.0.0.0] with about 10ml methanol (80/20, volume/volume) under 70 ℃ and 140bar pressure, 5 minutes heating phase and 1 minute static extraction extract polar material.With about 10ml ethanol/methylene (40/60, volume/volume) under 70 ℃ and 140bar pressure, 5 minutes heating phase and 1 minute static extraction extract more lipophilic material.Two kinds of solvent mixtures are extracted into (centrifuge tube that is used for ASE, 50ml have screw cap and can pierce through barrier film (DIONEX)) in the same Glass tubing.

[0505.0.0.0] is with interior mark treatment soln: ribitol, L-glycine-2,2-d ₂, L-L-Ala-2,3,3,3-d ₄, methionine(Met)-methyl-d ₃With Alpha-Methyl glucopyranoside and Nonadecanoic acid methylester, undecanoic acid methyl esters, tridecanoic acid methyl esters, pentadecylic acid methyl esters, montanic acid methyl esters.

[0506.0.0.0] uses 8ml water treatment general extractive.Abandon the solid residue and the extraction tube of plant sample.

[0507.0.0.0] shakes extract, and under 1400g at least centrifugal 5-10 minute then, to quicken the separation of phase.Shift out 1ml supernatant methanol phase (" polar phase ", colourless) and be used for further GC analysis, shift out 1ml again and be used for the LC analysis.Abandon remaining methanol phase.Shift out 0.5ml organic phase (" lipid phase ", deep green) and be used for further GC analysis, shift out 0.5ml again and be used for the LC analysis.All parts that shift out use the infrared vacuum-evaporator of IR Dancer (Hettich) to be evaporated to drying.Top temperature in the evaporation step is no more than 40 ℃.Pressure in the device is not less than 10mbar.

D) lipid that is used for LC/MS or LC/MS/MS analysis is handled mutually

[0508.0.0.0] has been evaporated to dried lipid-soluble extract and absorbed by moving phase.Use gradient elution to carry out HPLC.

[0509.0.0.0] has been evaporated to dried polar extract and absorbed by moving phase.Use gradient elution to carry out HPLC.

E) be used for the lipid phase derivatize that GC/MS analyzes

[0510.0.0.0] decomposes transferance (transmethanolysis) for methyl, to the mixture that adds 140 μ l chloroforms, 37 μ l hydrochloric acid (HCl is 37% by weight in the water), 320 μ l methyl alcohol and 20 μ l toluene in the extract of pervaporation.To manage envelope tightly and in 100 ℃ of vibrations heated 2 hours.Then solution evaporation is extremely done.With the residue complete drying.

[0511.0.0.0] carries out the amination of carbonyl methoxy in the tight Guan Zhongyu methoxamine hydrochloride of envelope (with the concentration of 5mg/ml, 100 μ l continue 1.5 hours in 60 ℃ in pyridine) reaction.Add 20 μ l odd number straight chain fatty acid solution (in 3/7 (volume/volume) pyridine/toluene, the solution of the lipid acid of the lipid acid of the 7-25-of an every kind of 0.3mg/mL carbon atom and 27,29 and 31 carbon atoms of every kind of 0.6mg/mL) according to time standard.At last, in the tight pipe of envelope, in 60 ℃ with 100 μ l N-methyl-N-(TMS)-2,2,2-trifluoroacetamide (MSTFA) derivatize 30 minutes.Being injected into GC final volume before is 220 μ l.

F) be used for the derivatize of the polar phase that GC/MS analyzes

[0512.0.0.0] carries out the amination of carbonyl methoxy in the tight Guan Zhongyu methoxamine hydrochloride of envelope (with the concentration of 5mg/ml, 50 μ l continue 1.5 hours in 60 ℃ in pyridine) reaction.Add 10 μ l odd number straight chain fatty acid solution (in 3/7 (volume/volume) pyridine/toluene, the solution of the lipid acid of the lipid acid of the 7-25-of an every kind of 0.3mg/mL carbon atom and 27,29 and 31 carbon atoms of every kind of 0.6mg/mL) according to time standard.At last, in the tight pipe of envelope, in 60 ℃ with 50 μ l N-methyl-N-(TMS)-2,2,2-trifluoroacetamide (MSTFA) derivatize 30 minutes.Being injected into GC final volume before is 110 μ l.

G) analysis of various plants sample

[0513.0.0.0] sample is measured according to the independent series (being also referred to as sequence) of every group of 20 plant samples, and each sequence comprises at least 5 wild-type plants in contrast.The peak area of each analyte is divided by marking peak area in each.According to the plant fresh weight with data normalization.Therefore, with the numerical value that calculates corresponding wild-type control group data mean value divided by same sequence, thus relevant with the wild-type control group.The numerical value that obtains is called as weight ratio (ratio by WT), and they are comparable between sequence, and shows in the concentration of analyte in mutant and the wild-type contrast relative different how much.Carried out appropriate control before, self the plant metabolism component has been had no significant effect with proof carrier and conversion.Therefore, compare with wild-type, the change of expectation is to be caused by the introducing gene.

[0514.0.0.0] selects as another kind of, as people such as Geigenberger (Plant Cell ﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect amino acid in the ethanol extraction.

Table 1 below the result of different plant analyses is visible:

?ORF	Note	Metabolite	Minimum value	Maximum value	Method
						?YBL015W	Acetyl-CoA hydrolase	Methionine(Met)	??1.42	??2.16	??LC
?YER173W	Check position protein	Methionine(Met)	??1.35	??1.60	??GC
						?YLR375W	The picked-up of tRNA montage and branched-chain amino acid before participating in	Methionine(Met)	??1.27	??2.93	??LC+??GC
?YOR084W	The peroxysome lipase of supposing	Methionine(Met)	??3.18	??3.18	??GC

?b1829	Heat shock protein with protease activity	Methionine(Met)	??1.29	??3.73	?GC
						?b4232	Fructose-1	Methionine(Met)	??1.20	??1.21	?LC
?b0464	The transcription repression albumen of multiple medicines efflux pump (TetR/AcrR family)	Methionine(Met)	??1.35	??4.66	?GC
						?b1343	ATP-RNA-dependent helicase is stimulated by 23S rRNA	Methionine(Met)	??1.38	??1.51	?GC
?b2414	The subunit of cysteine synthase A and O-acetylserine sulfhydrylase A, the PLP dependent enzyme	Methionine(Met)	??1.37	??1.75	?LC
						?b2762	3 '-adenosine phosphate 5 '-phosphinylidyne sulfuric acid (PAPS) reductase enzyme	Methionine(Met)	??1.43	??1.69	?LC+?GC

[0515.0.0.0] the 3rd row have shown the metabolite of being analyzed/each fine chemicals.The 4th row and the 5th row have shown the metabolite of the being analyzed/ratio of each fine chemicals between transgenic plant and wild-type.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 6th row have been pointed out analytical procedure.

Prove that all results are significant during [0516.0.0.0] independent replicate analysis.

[0517.0.0.0] embodiment 14a: transform the rye grass plant by for example expressing from crossing of yeast saccharomyces cerevisiae, intestinal bacteria or plant or other biological institute of the present invention characterising polynucleotides

The seed that [0518.0.0.0] can use some different rye grass mutation is as the explant source that is used to transform, and comprises the commodity mutation Gunne or the mutation Affinity that can obtain from Svalof Weibull seeds company.Seed is used 1%Tween-201 minute successively, and deionized water and distilled water rinsing three times are used in the surface sterilization in 60 minutes of 100% SYNTHETIC OPTICAL WHITNER, each 5 minutes, sprouts 3-4 days in dark on the aseptic filter paper of humidity then.And then with 1%Tween-20 1 minute, the sterilization in 5 minutes of 75% SYNTHETIC OPTICAL WHITNER was with ddH2O washing three times, each 5 minutes.

[0519.0.0.0] places the seed of surface sterilization on the callus inducing medium that contains Murashige and Skoog basis salinity and VITAMIN, 20g/l sucrose, 150mg/l l-asparagine, 500mg/l casein hydrolysate, 3g/lPhytagel, 10mg/l BAP and 5mg/l dicamba 98.Hatch 4 weeks of culture dish in 25 ℃ in the dark, so that the callus that seed germination and inducing embryo take place.

After 4 weeks on the callus inducing medium, the bud of stripling and root move to callus on the fresh culture [0520.0.0.0], around continuing to cultivate, are transferred to MSO substratum two weeks under light then again.Several pieces callus (11-17 age in week) are filtered with 10 order mesh screens, place then on the callus inducing medium, perhaps the 100mL liquid rye grass callus inducing medium (identical with the substratum that contains agar that is used for callus induction) in the 250mL triangular flask is cultivated.Triangular flask is wrapped with paper tinsel and was in the dark shaken for 1 week in 23 ℃ with 175 rev/mins.With 40 purpose mesh screens screening liquid culture and collecting cell.Be laid on the solid black wheat straw callus inducing medium at the composition of collecting on the screen cloth, and cultivated for 1 week in 25 ℃ in the dark.Then callus is transferred on the MS substratum that contains 1% sucrose and cultivated for 2 weeks.

[0521.0.0.0] can finish conversion by Agrobacterium or particle bombardment method.The expression vector of preparation comprises constitutive plant promoters and the gene cDNA in the pUC carrier.Use the Qiagen test kit and from Bacillus coli cells, prepare plasmid DNA according to manufacturer's specification sheets.The about 2g embryo callus of aseptic filter paper central authorities coating in culture dish.The aliquots containig that on filter paper, adds the liquid MSO that contains 10g/l sucrose.According to Sanford etc., 1993 method is wrapped up gold grain (1.0 μ m size) and by following parameter it is delivered to embryo callus with plasmid DNA: bombard 500 μ g particles and 2 μ g DNA, 1300psi at every turn, coil the range of callus dish from prevention from bombarding once for 8.5cm, every dish callus.

After [0522.0.0.0] bombardment, callus is shifted back in fresh healing tissue development's substratum, and room temperature keeps 1 time-of-week in the dark.Then callus is transferred to the following 25 ℃ growth conditions of light, to break up the down initial embryo of suitable selective agent (as 250nM Arsenal, 5mg/l PPT or 50mg/L kantlex).Appearance is just transferred in the soil in case take root the bud that selective agent has resistance.

[0523.0.0.0] is by sample the existing with confirmation T-DNA of the initial transgenic plant of pcr analysis (T0).These results confirm by Southern hybridization, wherein DNA electrophoresis and being transferred on the positively charged nylon membrane (Roche Diagnostics) in 1% sepharose.Use PCR DIG probe synthetic agent box (Roche Diagnostics) and prepare the probe of digoxigenin labeled according to manufacturer's recommendation by PCR.

[0524.0.0.0] transgenosis T0 rye grass plant tillers by excision and nourishes and generates.Tillering of transplanting kept two months until good foundation in the greenhouse.Remove bud and it was grown for two weeks.

[0525.0.0.0] embodiment 14b: the soybean plants of transforming by the expression that for example derives from yeast saccharomyces cerevisiae, intestinal bacteria or plant or other biological institute of the present invention characterising polynucleotides

[0526.0.0.0] is according to Texas A ﹠amp; M patent US 5,164, the amending method soybean transformation of method described in 310.Some commodity soybean mutation can transform by this method.Usually transform with cultivar Jack (can obtain) from Illinois Seed Foundation.By in 70% (v/v) ethanol, soaking 6 minutes and having added to soak in the 25% commodity SYNTHETIC OPTICAL WHITNER (NaOCL) of 0.1% (v/v) Tween and seed sterilized in 20 minutes, use aseptic bi-distilled water rinsing 4 times then.By take off the seedling that radicle, plumular axis and cotyledon were bred seven days from each seedling.Then, the epicotyl that will have a slice cotyledon is transferred on the germination medium fresh in the culture dish, and hatches for 3 weeks in 25 ℃ under 16 hour photoperiod (about 100 μ E-m-2s-1).Downcut axil joint (the about 4mm of length) from 3-4 plant in age in week.Excision axil joint is also hatched in Agrobacterium LBA4404 culture.

[0527.0.0.0] described (the An for example of the many different binary vector system that is used for Plant Transformation, G.in Agrobacterium Protocols.Methods in Molecular Biology, the 44th volume, the 47-62 page or leaf, Garland KMA and MR Davey compile, Humana Press, Totowa, New Jersey).Much be based on the described pBIN19 carrier of Bevan (Nucleic Acid Research.1984.12:8711-8721), it comprises the gene expression in plants box, left side that these expression cassette both sides are the agrobacterium tumefaciens Ti-plasmids and right side boundary sequence.The gene expression in plants box is by at least two genomic constitutions, i.e. selectable marker gene and regulate the cDNA of character gene or the plant promoter that genomic dna is transcribed.As previously mentioned, can use the multiple choices marker gene, comprise the arabidopsis gene (United States Patent (USP) 57673666 and 6225105) of encoding mutant type acetohydroxy acid synthase (AHAS).Similarly, as mentioned above, multiple promotor can be used for regulating character gene and regulates, grows and regulate, organize adjusting or environment to regulate with the composing type that genetic transcription is provided.In the present embodiment, use 34S promotor (GenBank accession number M59930 and X16673) so that the constitutive expression of character gene to be provided.

After [0528.0.0.0] co-cultivation was handled, the washing explant also was transferred to the selection substratum that has added 500mg/L Ticarcillin/Clavulanate Acid (Timentin).The cutting-out bud also places on the bud elongation medium.The bud of being longer than 1cm advances soil in transplanting and is prepended to 2-4 week on the root media.

[0529.0.0.0] initial transgenic plant (T0) by pcr analysis to confirm existing of T-DNA.These results confirm by Southern hybridization, wherein DNA electrophoresis and being transferred on the positively charged nylon membrane (Roche Diagnostics) in 1% sepharose.Use PCR DIG probe synthetic agent box (Roche Diagnostics) and prepare the probe of digoxigenin labeled according to manufacturer's recommendation by PCR.

[0530.0.0.0] embodiment 14c: transform maize plant by the expression of crossing that for example derives from yeast saccharomyces cerevisiae, intestinal bacteria or plant or other biological institute of the present invention characterising polynucleotides.

[0530.1.0.0] following 5 except upstream primer SEQ ID NO:3 and reverse primer SEQ ID NO:4 contain ' extending, as carrying out the amplification of SEQ ID NO:1 described in the embodiment 10:

I) forward primer: 5 '-GGGTCGCTCCTACGCG-3 ' SEQ ID NO:68243

Ii) reverse primer: 5 '-CTCGGGCTCGGCGTCC-3 ' SEQ ID NO:68246

[0530.2.0.0]: vector construction

The following structure of corn conversion carrier that is used for constitutive expression.

Select carrier EG073qcz (SEQ ID NO 68240) and EG065qcz (SEQ ID NO:68241) as underlying carrier.By delete MCS with Asp718 and PstI digested vector, use the T4DNA polysaccharase subsequently with the carrier flush endization from EG065qcz.The carrier of flush endization is reconnected.The carrier that is produced is called EG065-MCS.Produce dna fragmentation by hybridizing following oligonucleotide, the LIC box is cloned into carrier EG065-MCS with the end in the carrier that can connect into SmaI and SacI digestion.This fragment is connected into the carrier EG065-MCS that has digested with SmaI and SacI.The carrier that is produced is called EG065-LIC.Downcut from EG065-LIC with AscI and PacI and to comprise ScBV (Schenk (1999) Plant Mol Biol39 (6): 1221-1230) the expressed intact box of promotor, LIC box and terminator, and connecting among the previous carrier EG073qcz with AscI and PacI digestion.The resulting binary vector that is used for the corn conversion is called pMME0607 (SEQ ID NO:68242).

Oligonucleotide POCCLicMluISacIIfw:

gggtcgctcctacgcgtcaatgatccgcggacgccgagcccgagct(SEQ?ID?NO：68244)

Oligonucleotide POCCLicMluISacIrev:

cgggctcggcgtccgcggatcattgacgcgtaggagcgaccc(SEQ?ID?NO：68245)

For clone polynucleotide of the present invention (for example ORF of SEQ ID NO:1) from yeast saccharomyces cerevisiae, handle carrier DNA with restriction enzyme MluI and SacII.Come termination reaction by inactivation, and go up purifying at QIAquick post (Qiagen) according to standard program at 70 ℃, 20 minutes.

Use the T4DNA polysaccharase then and handle PCR product and the carrier DNA represent ORF that increases according to standard program (MBI Fermentas), so that the generation strand overhangs, for the reaction parameter of carrier be: 1 T4 of unit archaeal dna polymerase, 37 ℃ reacted 2-10 minute; Reaction parameter for the PCR product of representing SEQID NO:1 is: 1u T4 archaeal dna polymerase, 15 ℃ the reaction 10-60 minute.

Add the high-salt buffer termination reaction and carry out purifying according to standard program (Qiagen) on the QIAquick post.

Carrier that about 30ng is prepared and true quantitative prepared amplified material mix and are incorporated in 65 ℃ of hybridization 15 minutes, reduce to 37 ℃ with 0.1 ℃/1 second subsequently, keep 10 minutes at 37 ℃ then, reduce to 4 ℃ with 0.1 ℃/1 second then.

Add competence Bacillus coli cells (DH5 α bacterial strain) and hatched 20 minutes at 1 ℃ in same reactor, 42 ℃ of heat-shockeds 90 seconds and be cooled to 4 ℃ connect the construct conversion with institute subsequently.Then, add perfect medium (SOC) and mixture hatched 45 minutes at 37 ℃.Whole mixtures are coated on the agar plate that contains the 0.05mg/ml kantlex and subsequently 37 ℃ of overnight incubation.

Utilization is increased in conjunction with the primer of integration site upstream and downstream, thereby allows the amplification insertion portion, verifies clone result.Described in the operation steps of Taq archaeal dna polymerase (Gibco-BRL), increase.

Amplification cycles is as follows: 94 ℃, and 5 minutes, a circulation; 94 ℃ subsequently, 15 seconds, 50-66 ℃, 15 seconds and 72 ℃, 5 minutes, 35 circulations; 72 ℃ subsequently, 10 minutes, 1 circulation; Place 4 ℃ then.

Detected several bacterium colonies, but a bacterium colony that detects the big or small PCR product of expection is used for following steps:

This positive bacterium colony partly is transferred in the reaction vessel that contains the perfect medium (LB) that adds kantlex and 37 ℃ of overnight incubation.

Such as in Qiaprep (Qiagen) normal process detailed description carry out plasmid preparation.

[0530.3.0.0] embodiment 14c.a.: the conversion of corn

The preparation of immature embryo and Agrobacterium is basically as US 5,591, described in 616.In brief, the agrobacterium strains LBA4404 that uses plasmid to transform by standard method (as triple crossing method or electroporation) grew 2 days on the LB plate, carried out common cultivation subsequently.One garland cells is resuspended in liquid with about 1.0 O.D. to be infected in the substratum.The immature embryo of about 1.5mm size was hatched in Agrobacterium solution about 30 minutes.From liquid, take out and exsomatize embryo and in 22 ℃ of dark, (comprise 2mg/l 2,4-D, 10 μ m AgNO at callus induction solid medium based on MS with agrobacterium tumefaciens ₃With 200 μ m Syringylethanones) go up altogether and cultivate.After cultivating several days altogether, the substratum that embryo is transferred to based on MS (comprises 2mg/l 2,4-D, 10 μ m AgNO ₃With the 200mg/l Ticarcillin/Clavulanate Acid) cultivated for 1 week in 27 ℃ in the dark.With embryo be transferred to based on MS, comprise tetrahydroglyoxaline weedicide (500nM Pu Shite (Pursuit)) as 3 weeks of dark culturing in the selection substratum of selective agent.After 3 weeks, supposition transgenosis embryo is transferred to the substratum (comprising 2mg/L kinetin, 500nM Pu Shite and 200mg/l Ticarcillin/Clavulanate Acid) based on MS and under the white luminescent lamp of cool colour (100uE/m2/s-1, the photoperiod is 16 hours), cultivates 2-3 week or form up to bud in 25 ℃.Bud is transferred to based on the root media of MS and in 25 ℃ of 2 weeks of illumination cultivation.The bud that to take root be implanted into 4 inches contain the artificial soil mixture the jar in.Add 360 and in indoor growth 1-2 week of artificial environment.The environmental cabinet remains on illumination in 16 hours and the 8 hours dark diurnal cycle under 27 ℃ and 22 ℃ respectively.Mix working strength and provide illumination for warm colour and the white luminescent lamp of cool colour of～400uE/m2/s-1.Plant-growth is transferred to them to the 4-6 leaf phase and comprises

In 360 14 inches the jar.Increase to use metal-halide lamp with keep intensity＞800uE/m2/s-1 and 28 ℃ of illumination in 16 hours on daytime and night 22 ℃ of diurnal cycle of 8 hours.Transplant on every Tuesdays.After carrying out the T0 sampling, Monday weekly and Thursday twice use Peters 20-20-20 adds micro-nutrients (200ppm) and applies fertilizer to plant.Produce the T1 seed from performance tolerance imidazolidinone weedicide and transgenosis PCR male plant.Term single gene seat with T-DNA inserts (selfing) T0 plant and produces T1 generation, and T1 separates with 3: 1 ratios for transgenosis.The filial generation that contains transgenosis copy can tolerate imidazolidinone weedicide and can detect by pcr analysis.

The growth of [0530.4.0.0] embodiment 14c.b.:T0 maize plant is used for metabolite analysis

Plant suitable stage of growing under following normalization condition is used for T0 sampling.After the T0 plantlet grows to the 4-6 leaf phase (1-3 week), they are transferred to 14 in the greenhouse " jar in.Follow of the contrast of the plant that contains pBSMM232 of each experiment generation as the T0 sample.On Tuesday weekly plantlet is transplanted to 14 " jar in.Plant strain growth 9 days is up to reaching the 7-13 leaf phase.Between at 10 in the morning and 2 pm on Thursday, go up sampling at the 3rd lobus cardiacus (the 1st full extension leaf).Taking off 250-500mg leaf material (not containing middle arteries and veins) in 30 seconds weighs and is positioned in the preextraction glass bushing in the liquid nitrogen.The sample that to obtain from the every strain plant offside of arteries and veins () sample preparation is used for aforesaid qPCR and analyzes.

[0530.5.0.0] embodiment 14c.c.: be used for the T1 maize plant growth of metabolite analysis

For the bioanalysis of transgenic plant, latter's homogeneous grows in the specific culture apparatus.For this reason, the GS-90 substrate is joined body maker device (Laible System GmbH, Singen, Germany) as compost mixture and be added in the jar.After this, 26 jars are combined in the dish and with Pu Like handle.For processing, add in the 10l tap water that 25ml is general to be made every effort to overcome.This amount is enough to handle about 150 jars.The tap water that jar is positioned in the Pu Like solution and other usefulness does not contain Pu Like is irrigated on top.They used in 4 days.

For sowing, taking-up has been stored in the seed of room temperature and has been transferred in the jar with soil from paper bag.About altogether 1-3 grain seed is distributed in the centre of jar.

After the sowing seed, placed 2 days with the plate of the plastic lousing covering loading jar that is complementary and in the growth room.Remove plastic lousing after 2 days and be positioned over plant on the growth platform and under following growth conditions, cultivated 22-24 days: in 20 ℃ of illumination in 16 hours, 8 hours dark cycle, 60% atmospheric moisture and the about CO of 400ppm ₂Concentration.Employed light source is the PowerstarHQI-T 250W/D fluorescent lamp from Osram, and what it produced about 220 μ E/m2/s-1 light intensities is similar to sun stratographic light.

When plant strain growth in the time of 7 days, they are carried out the transgenic plant screening.For this reason, the fragment with plant leaf carries out sample preparation and uses genetically modified primer separately to carry out the PCR reaction.Present genetically modified plant and be used for metabolic analysis.Remove not genetically modified seedling.When transfer-gen plant reaches 18 day age, with they thinnings.Grow best transfer-gen plant as the target plant in jar central authorities.Take out all the residue plant and abandon by means of the metal tweezers are careful.

During plant strain growth, irrigate in the placement groove in top lavation (on compost) and from the bottom with distilled water.In case growth plant reaches 24 day age, the results plant.

[0530.6.0.0] embodiment 14c.d.: the metabolic analysis of leaf of Semen Maydis.

By the modification of following steps evaluation according to the inventive method evaluation, above-mentioned metabolite content.

A) sample preparation of sample and storage

In near the corridor in greenhouse, carry out sample preparation.Use little laboratory scissors to shear leaf for twice and also therefrom take off this part blade on the arteries and veins by hand.Rapid weighing sample and be transferred to it in extraction sleeve pipe of precooling and be positioned in the ice chest of cooled with liquid nitrogen on the scale of laboratory.Shearing blade was no more than for 30 seconds to being frozen in the time total that consumes between the liquid nitrogen.Box is stored in-80 ℃ of refrigerators, on dry ice, transports.

B) freeze-drying

Experimental session carefully makes plant remain in deep refrigeration state (temperature＜-40 ℃) or anhydrous by freeze-drying, up to the solvent contact first time.Carry out before the analytic process, the extraction sleeve pipe that sample is housed is transferred to the aluminium frame of precooling.

Being mounted with the aluminium frame that is positioned over the plant sample in the extraction sleeve pipe is positioned in (40 ℃) freeze-drying apparatus of precooling.Starting temperature in the main drying stage is 0.120mbar for-35 ℃ and pressure.Between dry epoch, according to pressure and temperature routine change parameter.Outlet temperature after 12 hours is 0.001-0.004mbar for+30 ℃ and resulting pressure.After turning off vacuum pump and refrigerator, with air (via the drying tube exsiccant) or argon cleaning system.

C) extract

After the flushing low pressure moisture eliminator, the extraction sleeve pipe that the lyophilized vegetable material will be housed immediately is transferred to the 5ml extraction tube interior (an accelerated solvent extractor ASE 200 (DIONEX) who has solvent controller and AutoASE software) of ASE device.

Fill 24 sample positions of full ASE device (the accelerated solvent extractor ASE 200 (DIONEX) that has solvent controller and AutoASE software) with plant sample (comprising some samples that are used to test Quality Control).

With about 10ml methanol (80/20, volume/volume) under 70 ℃ and 140bar pressure, 5 minutes heating phase and 1 minute static extraction extract polar material.With about 10ml ethanol/methylene (40/60, volume/volume) under 70 ℃ and 140bar pressure, 5 minutes heating phase and 1 minute static extraction extract more lipophilic material.Two kinds of solvent mixtures are extracted into (centrifuge tube that is used for ASE, 50ml have screw cap and can pierce through barrier film (DIONEX)) in the same Glass tubing.

With interior mark treatment soln: ribitol, L-glycine-2,2-d ₂, L-L-Ala-2,3,3,3-d ₄, methionine(Met)-methyl-d ₃With Alpha-Methyl glucopyranoside and Nonadecanoic acid methylester, undecanoic acid methyl esters, tridecanoic acid methyl esters, pentadecylic acid methyl esters, montanic acid methyl esters.

With 8ml water treatment general extractive.Abandon the solid residue and the extraction tube of plant sample.

Shake extract, under 1400g at least centrifugal 5-10 minute then, to quicken the separation of phase.Shift out 0.5ml supernatant methanol phase (" polar phase ", colourless) and be used for further GC analysis, shift out 0.5ml again and be used for the LC analysis.All the remainder of the methanol phase of sample is used for extra quality control.Shift out 0.5ml organic phase (" lipid phase ", deep green) and be used for further GC analysis, shift out 0.5ml again and be used for the LC analysis.All parts that shift out use the infrared vacuum-evaporator of IR Dancer (Hettich) to be evaporated to drying.Top temperature in the evaporation step is no more than 40 ℃.Pressure in the device is not less than 10mbar.

D) lipid that is used for LC/MS or LC/MS/MS analysis is handled mutually

Being evaporated to dried lipid-soluble extract is absorbed by moving phase.Use gradient elution to carry out HPLC.

Being evaporated to dried polar extract is absorbed by moving phase.Use gradient elution to carry out HPLC.

E) be used for the lipid phase derivatize that GC/MS analyzes

Decompose transferance for methyl, to the mixture that in the extract of pervaporation, adds 140 μ l chloroforms, 37 μ l hydrochloric acid (HCl is 37% by weight in the water), 320 μ l methyl alcohol and 20 μ l toluene.To manage envelope tightly and in 100 ℃ of vibrations heated 2 hours.Then solution evaporation is extremely done.With the residue complete drying.

Carry out the amination of carbonyl methoxy in the tight Guan Zhongyu methoxamine hydrochloride of envelope (with the concentration of 20mg/ml, 100 μ l continue 1.5 hours in 60 ℃ in pyridine) reaction.Add 20 μ l odd number straight chain fatty acid solution (in 3/7 (volume/volume) pyridine/toluene, the solution of the lipid acid of the lipid acid of the 7-25-of an every kind of 0.3mg/mL carbon atom and 27,29 and 31 carbon atoms of every kind of 0.6mg/mL) according to time standard.At last, in the tight pipe of envelope, in 60 ℃ with 100 μ l N-methyl-N-(TMS)-2,2,2-trifluoroacetamide (MSTFA) derivatize 30 minutes.Being injected into GC final volume before is 220 μ l.

F) be used for the derivatize of the polar phase that GC/MS analyzes

Carry out the amination of carbonyl methoxy in the tight Guan Zhongyu methoxamine hydrochloride of envelope (with the concentration of 20mg/ml, 50 μ l continue 1.5 hours in 60 ℃ in pyridine) reaction.Add 10 μ l odd number straight chain fatty acid solution (in 3/7 (volume/volume) pyridine/toluene, the solution of the lipid acid of the lipid acid of the 7-25-of an every kind of 0.3mg/mL carbon atom and 27,29 and 31 carbon atoms of every kind of 0.6mg/mL) according to time standard.At last, in the tight pipe of envelope, in 60 ℃ with 50 μ l N-methyl-N-(TMS)-2,2,2-trifluoroacetamide (MSTFA) derivatize 30 minutes.Being injected into GC final volume before is 110 μ l.

G) analysis of various plants sample

Sample is measured according to the independent series (being also referred to as sequence) of 20 every group plants (leaf) sample, and each sequence comprises at least 5 samples that come the different adjoining trees of self-contained GUS.The peak area of each analyte is divided by marking peak area in each.Gather in the crops sample fresh weight with data normalization according to each.Therefore, with the numerical value that calculates corresponding control group data mean value divided by same sequence, thus relevant with the control group that contains GUS.The numerical value that obtains is called as weight ratio, and they are comparable between sequence, and shows in the concentration of analyte in mutant and the wild-type contrast relative different how much.Selection contains the plant of GUS and so that guarantee carrier and conversion operation self metabolite component in the plant is had no significant effect.Therefore, compared with the control, the change of expectation is to be caused by the introducing gene.

The amending method of (1996.Nature Biotech14745-50) described methods such as [0531.0.0.0] use Ishida also can transform Zea mays.In corn, conversion is that genotype is dependent and have only the specific genes type just to can be used for transforming and regeneration.Self-mating system A188 (University of Minnesota) or be to transform the good source of donor material (Fromm etc., 1990Biotech8:833-839), but other genotype also can successfully be used as parent's hybrid with A188.Fringe when immature embryo about 1 arrives 1.2mm, is gathered from maize plant in about 11 days of pollination back (DAP).With the immature embryo and the agrobacterium tumefaciens co-cultivation of carrying " super binary " carrier, and come the regeneration of transgenic plant by organ.Super binary vector system description to some extent in WO patent WO 94/00977 and WO 95/06722 of Japan Tobacco (Japan Tobacco).Carrier construction as described.Can use the multiple choices marker gene, comprise the corn gene of encoding mutant type acetohydroxy acid synthase (AHAS) (United States Patent (USP) 6025541).Similarly, can use multiple promotor to regulate character gene, regulate, grow and regulate, organize adjusting or environment to regulate with the composing type that genetic transcription is provided.In the present embodiment, use 34S promotor (GenBank accession number M59930 and X16673) that the constitutive expression of character gene is provided.

[0532.0.0.0] be the culture of ex vivo embryo on callus inducing medium, cultivates on the corn regeneration culture medium that contains as the imidazolone of selective agent then.Culture dish is cultivated 2-3 weeks in 25 ℃ under light, or grows up to bud.The bud of green is moved to the maize rooting substratum and cultivates 2-3 week up to root development at 25 ℃ from each embryo.The bud that to take root is transplanted in the greenhouse soil.From imidazolidinone weedicide being shown patience and PCR detect to producing the T1 seed the plant of transgenic positive.

The T1 that [0533.0.0.0] T-DNA term single gene seat inserts separates transgenosis with 3: 1 ratio from generation to generation.The offspring who comprises one or two transgenosis copy tolerates imidazolidinone weedicide.The T2 plant of isozygotying shows the phenotype similar to the T1 plant.The hybrid plant (F1 offspring) of transgenic plant and non-transgenic plant of isozygotying also demonstrates the similar phenotype of raising.

[0534.0.0.0] embodiment 14d: transform wheat plant by the expression of crossing that for example derives from yeast saccharomyces cerevisiae, intestinal bacteria or plant or other biological institute of the present invention characterising polynucleotides

[0535.0.0.0] transforms by carry out wheat as (1996Nature Biotech.14745-50) described methods such as Ishida.Usually use cultivar Bobwhite (can obtain) to transform from Mexican CYMMIT.With immature embryo be loaded with the agrobacterium tumefaciens co-cultivation of " super binary " carrier and by organ generation regeneration of transgenic plant.Super binary vector system description to some extent in WO patent WO 94/00977 and WO 95/06722 of Japan Tobacco (Japan Tobacco).Carrier construction as described.Can use the multiple choices marker gene, comprise the corn gene of encoding mutant type acetohydroxy acid synthase (AHAS) (United States Patent (USP) 6025541).Similarly, can use multiple promotor to regulate character gene, regulate, grow and regulate, organize adjusting or environment to regulate with the composing type that genetic transcription is provided.Use 34S promotor (GenBank accession number M59930 and X16673) so that the constitutive expression of character gene to be provided.

After Agrobacterium hatched, embryo was cultivated on callus inducing medium [0536.0.0.0], cultivated on the regeneration culture medium that contains as the imidazolone of selective agent then.Culture dish is cultivated 2-3 weeks in 25 ℃ under light, or grows up to bud.The bud of green is moved to root media and cultivates 2-3 week up to root development at 25 ℃ from each embryo.The bud that to take root is transplanted in the greenhouse soil.From imidazolidinone weedicide being shown patience and PCR detect to producing the T1 seed the plant of transgenic positive.

The T1 that [0537.0.0.0] T-DNA term single gene seat inserts separates transgenosis with 3: 1 ratio from generation to generation.The offspring who comprises one or two transgenosis copy tolerates imidazolidinone weedicide.The T2 plant of isozygotying shows similar phenotype.

[0538.0.0.0] embodiment 14e: transform Semen Brassicae campestris/rape plant by the expression of crossing that for example derives from yeast saccharomyces cerevisiae, intestinal bacteria or plant or other biological institute of the present invention characterising polynucleotides

[0539.0.0.0] uses the cotyledon petiole of seedling in 5-6 days ages and the hypocotyl explant as tissue culture, and transforms according to (1998, Plant Cell Rep 17:183-188) such as Babic.Commodity cultivar Westar (Agriculture Canada) is the standard mutation that is used to transform, but can use other mutation.

[0540.0.0.0] uses the agrobacterium tumefaciens LBA4404 that contains binary vector to transform rape.(the An for example of many different binary vector systems that are used for Plant Transformation has been described, G.inAgrobacterium Protocols.Methods in Molecular Biology 44 volumes, the 47-62 page or leaf, Garland KMA and MR Davey compile, Humana Press, Totowa, New Jersey).Much be based on the described pBIN19 carrier of Bevan (Nucleic Acid Research.1984.12:8711-8721), it comprises the gene expression in plants box, and the expression cassette both sides are left side and the right side boundary sequence that derives from the agrobacterium tumefaciens Ti-plasmids.The gene expression in plants box is by at least two genomic constitutions, i.e. selectable marker gene and regulate the cDNA of character gene or the plant promoter that genomic dna is transcribed.Can use the multiple choices marker gene, comprise the arabidopsis gene of encoding mutant type acetohydroxy acid synthase (AHAS) (United States Patent (USP) 57673666 and 6225105).Similarly, multiple promotor can be used for regulating character gene, regulates, grows and regulate, organize adjusting or environment to regulate with the composing type that genetic transcription is provided.Use 34S promotor (GenBank accession number M59930 and X16673) so that the constitutive expression of character gene to be provided.

[0541.0.0.0] soaks 10 minutes with the Semen Brassicae campestris surface sterilization then by soaking two minutes in the 30%Clorox that contains a Tween-20 in 70% ethanol, use aseptic distilled water rinsing three times subsequently.Make seed external sprouting 5 days under illumination in 23 ℃, 16 hours in the half intensity MS substratum that does not contain hormone, 1% sucrose, 0.7%Phytagar then.Downcut the cotyledon petiole explant that has cotyledon from external seedling, and inoculate Agrobacterium by excision end immersion bacterial suspension with the handle explant.Then explant was being cultivated two days under illumination in 23 ℃, 16 hours on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7%Phytagar.With the Agrobacterium co-cultivation two days later, petiole explant moved on the MSBAP-3 substratum that contains 3mg/l BAP, cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid (300mg/l) and cultivated 7 days, cultivate up to shoot regeneration containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent then.When the long 5-10mm of bud with its cutting-out and move on the bud elongation medium (MSBAP-0.5 that contains 0.5mg/l BAP).The bud that is about 2cm moved on the root media (MS0) carry out root induction.

The sample of [0542.0.0.0] original transgenic plant (T0) is confirmed the existence of T-DNA by pcr analysis.These results confirm by Southern hybridization, wherein DNA electrophoresis and being transferred on the positively charged nylon membrane (Roche Diagnostics) in 1% sepharose.Use PCR DIG probe synthetic agent box (Roche Diagnostics) and prepare the probe of digoxigenin labeled according to manufacturer's recommendation by PCR.

[0543.0.0.0] embodiment 14f: transform the alfalfa plant by the expression of crossing that for example derives from yeast saccharomyces cerevisiae, intestinal bacteria or plant or other biological institute of the present invention characterising polynucleotides

The method of (1999 Plant Physiol 119:839-847) such as [0544.0.0.0] use McKersie transforms the regeneration clone of alfalfa (alfalfa).The regeneration of alfalfa and conversion are that genotype is dependent, therefore need aftergrowth.Obtain existing description of method of aftergrowth.For example, they can be selected from Lan Jilande (Rangelander) cultivar (Agriculture Canada) or as BrownDCW and described any other commodity alfalfa mutation of A Atanassov (1985.Plant Cell Tissue Organ Culture 4:111-112).In addition, can select RA3 mutation (winconsin university) to be used for tissue culture (Walker etc., 1978 Am J Bot 65:654-659).

[0545.0.0.0] is with petiole explant and agrobacterium tumefaciens C58C1pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or contain the overnight culture co-cultivation of the LBA4404 of binary vector.(the An for example of many different binary vector systems that are used for Plant Transformation has been described, G.Agrobacterium Protocols. " Methods in Molecular Biology " the 44th volume, the 47-62 page or leaf, Garland KMA and MR Davey compile, Humana Press, Totowa, New Jersey).Much be based on the described pBIN19 carrier of Bevan (Nucleic Acid Research.1984.12:8711-8721), it comprises the gene expression in plants box that both sides and the left side that derives from the agrobacterium tumefaciens Ti-plasmids link to each other with the right side boundary sequence.The gene expression in plants box is by at least two genomic constitutions, i.e. selectable marker gene and regulate the cDNA of character gene or the plant promoter that genomic dna is transcribed.Can use the multiple choices marker gene, comprise the arabidopsis gene of the acetohydroxy acid synthase (AHAS) (United States Patent (USP) 57673666 and 6225105) of encoding mutant.Similarly, multiple promotor can be used for regulating character gene, regulates, grows and regulate, organize adjusting or environment to regulate with the composing type that genetic transcription is provided.Use 34S promotor (GenBank accession number M59930 and X16673) so that the constitutive expression of character gene to be provided.

[0546.0.0.0] explant is containing 288mg/L proline(Pro), 53mg/L Thioproline, 4.35g/L K ₂SO ₄And on the SH inducing culture of 100 μ M Syringylethanones in dark place co-cultivation three days.Washing explant and be layered on and do not contain Syringylethanone and contain suitable selective agent and suppress on the suitable antibiotic identical SH inducing culture of Agrobacterium growth in the half intensity Murashige-Skoog substratum (Murashige and Skoog, 1962).After several weeks, somatic embryo is moved to the BOi2Y that does not contain growth regulatory substance and microbiotic and contain 50g/L sucrose grow in the substratum.Somatic embryo is grown on half intensity Murashige-Skoog substratum subsequently.The seedling that to take root is transplanted in the basin and in the greenhouse and is cultivated.

[0547.0.0.0] taken root by eminectomy and in the Turface growth medium and bred the T0 transgenic plant.Remove the leaf of plant and be cultured to the height (about two weeks after the disleaf) of about 10cm.

[0548.0.0.0] embodiment 14g: transform the alfalfa plant by the expression of crossing that for example derives from yeast saccharomyces cerevisiae, intestinal bacteria or plant or other biological institute of the present invention characterising polynucleotides

The method of (1999 Plant Physiol 119:839-847) such as [0549.0.0.0] use McKersie transforms the regeneration clone of alfalfa (alfalfa).The regeneration of alfalfa and conversion are that genotype is dependent, therefore need aftergrowth.Obtain existing description of method of aftergrowth.For example, they can be selected from Lan Jilande (Rangelander) cultivar (Agriculture Canada) or as BrownDCW and described any other commodity alfalfa mutation of A Atanassov (1985.Plant Cell Tissue Organ Culture 4:111-112).In addition, can select RA3 mutation (winconsin university) to be used for tissue culture (Walker etc., 1978 Am J Bot 65:654-659).

[0550.0.0.0] is with petiole explant and agrobacterium tumefaciens C58C1 pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or contain the overnight culture co-cultivation of the LBA4404 of binary vector.(the An for example of many different binary vector systems that are used for Plant Transformation has been described, G.Agrobacterium Protocols. " Methods in Molecular Biology " the 44th volume, the 47-62 page or leaf, Garland KMA and MR Davey compile, Humana Press, Totowa, New Jersey).Much be based on the described pBIN19 carrier of Bevan (Nucleic Acid Research.1984.12:8711-8721), it comprises the gene expression in plants box that both sides and the left side that derives from the agrobacterium tumefaciens Ti-plasmids link to each other with the right side boundary sequence.The gene expression in plants box is by at least two genomic constitutions, i.e. selectable marker gene and regulate the cDNA of character gene or the plant promoter that genomic dna is transcribed.Can use the multiple choices marker gene, comprise the arabidopsis gene of the acetohydroxy acid synthase (AHAS) (United States Patent (USP) 57673666 and 6225105) of encoding mutant.Similarly, multiple promotor can be used for regulating character gene, regulates, grows and regulate, organize adjusting or environment to regulate with the composing type that genetic transcription is provided.In the present embodiment, use 34S promotor (GenBank accession number M59930 and X16673) so that the constitutive expression of character gene to be provided.

[0551.0.0.0] explant is containing 288mg/L proline(Pro), 53mg/L Thioproline, 4.35g/L K ₂SO ₄And on the SH inducing culture of 100 μ M Syringylethanones in dark place co-cultivation three days.Washing explant and be layered on and do not contain Syringylethanone and contain suitable selective agent and suppress on the suitable antibiotic identical SH inducing culture of Agrobacterium growth in the half intensity Murashige-Skoog substratum (Murashige and Skoog, 1962).After several weeks, somatic embryo is moved to the BOi2Y that does not contain growth regulatory substance and microbiotic and contain 50g/L sucrose grow in the substratum.Somatic embryo is grown on half intensity Murashige-Skoog substratum subsequently.The seedling that to take root is transplanted in the basin and in the greenhouse and is cultivated.

[0552.0.0.0] taken root by eminectomy and in the Turface growth medium and bred the T0 transgenic plant.Remove the leaf of plant and be cultured to the height (about two weeks after the disleaf) of about 10cm.

[0552.1.0.0]./.

[0552.2.0.0] embodiment 16: prepare homologous sequence from plant

Grow under the standard that different plants can be in the greenhouse or the condition of change.RNA can extract according to Jones, Dunsmuir and Bedbrook (1985) EMBO method J.4:2411-2418.The organization material of about 1 gram from Different Organs ground in liquid nitrogen.With powder transfer to containing 4.5ml NTES damping fluid (100mM NaCl, 10mM Tris/HCl pH 7.5,1mMEDTA, 1%SDS; Be dissolved in the water of no RNA enzyme) and the 13ml Falcon pipe of 3ml phenol/chloroform/primary isoamyl alcohol (25/24/1) in, mixing and being stored on ice immediately.Use whizzer (Sorval; SM24 or SS34 rotor) with 7000 rev/mins with centrifugal 10 minutes of mixture.Supernatant is transferred in the new pipe, adds the 3M sodium-acetate (pH 5.2, are dissolved in the water of no RNA enzyme) of 1/10 volume and the Virahol of 1 times of volume, mixes the back and stores 1 hour or spend the night in-20 ℃.With 7000 rev/mins of centrifugal mixtures 10 minutes.Abandoning supernatant is also with 70% ethanol (v/v) washing precipitation.With 7000 rev/mins of centrifugal mixtures 5 minutes, abandoning supernatant also will precipitate dry air.Adding 1ml does not have the water of RNA enzyme and makes DNA/RNA be deposited in 4 ℃ of dissolvings on ice.Be transferred to nucleic acid solution in the 2ml Eppendorf pipe and add 1ml 4M Lithium Acetate.Solution can or spend the night in 4 ℃ of placements 3 hours at least behind the mixing.With 14000 rev/mins of centrifugal mixtures 10 minutes, abandoning supernatant also precipitated with 70% washing with alcohol, and dry air also is dissolved in 200 μ l and does not have in the water of RNA enzyme.

Total RNA can be used for method (for example using the synthetic and clone's test kit of ZAP-cDNA of Stratagene, La Jolla, the U.S.) the construction cDNA library according to manufacturers.Basically, use oligo (dT) joint primer to start the synthetic article one chain of messenger RNA(mRNA) (mRNA), and use reversed transcriptive enzyme to carry out reverse transcription.After the second chain cDNA is synthetic, double-stranded cDNA is connected into Uni-ZAP XR carrier.Uni-ZAP XR carrier allows pBluescript phagocytosis plastochondria inscribe to remove.The polylinker of pBluescript phagemid has 21 unique cloning sites, and its both sides are T3 and T7 promotor, and can select to be used for dna sequencing to 6 different primer sites.The systemic single operation order-checking terminal to 5 expection initiations of clone can allow for example by means of pedant pro software package (Biomax, M ü nchen) sequence to be carried out preliminary note.Retrieve the clone that can identify nucleic acid of the present invention or be used for the nucleic acid of the inventive method based on the homology of using canonical algorithm (as blastp or gap).The supposition full-length clone with identity or high homology that is identified is further checked order, so that obtain full sequence.

As mentioned above, by prepare each cDNA library from different plant origins, can identify the homologous sequence that other is new in a similar manner.Can use available sequences of the present invention under low stringency condition, to screen the library then, for example as Sambrook etc., described in the Molecular Cloning:Alaboratory manual, Cold Spring Harbor 1989, Cold Spring HarborLaboratory Press.The positive colony of purifying can be used for cutting technique and sufficient sequence analysis in the body.Use blastp or gap program can identify directly that to the paired sequence alignment of original series and new sequence to homologue (ortholog) (meaning is the homologous sequences from different biologies), it should have at least 30% sequence identity.In addition, the conservative property of function important amino acid residue or structural domain (can identify by comparing several paralogs that obtained) can be identified as new straight new sequence to homologue.

In addition, the library can be used for extensive sequencing and the sequence that obtained can be stored in the sequence library, can screen this sequence library by different searching algorithm (for example utilizing the tbastn algorithm of the nucleotide sequence that aminoacid sequence search of the present invention obtained) then and seek supposition directly to homologue.Clone with highest serial identity is used for full sequence and determines and directly can identify as mentioned above to homologue.

[0553.0.0.0]

1. produce the method for methionine(Met), it comprises:

(a) improve in non-human being or its one or more parts or produce as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation methionine(Met).

2. produce the method for methionine(Met), be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part methionine(Met) quantity to be increased;

B) contain just like Table I the 5th or 7 row, 1-5 is capable and/or the nucleic acid molecule of the nucleic acid molecule of 334-338 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of methionine(Met) quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of methionine(Met) quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that methionine(Met) quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 1-5 is capable and/or 334-338 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that each fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that methionine(Met) quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 1-5 is capable and/or 334-338 capable shown in consensus sequence polypeptide and give biology or its part in each fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give each fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded methionine(Met).

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded methionine(Met) of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part methionine(Met) quantity to be increased;

B) contain just like Table I the 5th or 7 row, 1-5 is capable and/or the nucleic acid molecule of the nucleic acid molecule of 334-338 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of methionine(Met) quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of methionine(Met) quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that methionine(Met) quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 1-5 is capable and/or 334-338 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that each fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that methionine(Met) quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 1-5 is capable and/or 334-338 capable shown in consensus sequence polypeptide and in biological or its part, give each fine chemicals quantity and increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give each fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt; Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, 1-5 is capable and/or 334-338 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 1-5 is capable and/or 334-338 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in methionine(Met) quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part methionine(Met) quantity increase coded by the nucleic acid molecule of claim 5 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps methionine level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the methionine level that will measure or expression of polypeptides level and standard methionine(Met) or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the methionine(Met) generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of methionine(Met) quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of methionine(Met) quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the methionine(Met) generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that methionine(Met) increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce methionine(Met);

(d) in host cell, express the nucleic acid molecule of being identified;

(e) methionine level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give methionine level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the methionine(Met) generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that methionine(Met) quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce methionine(Met);

(c) in host cell, express the nucleic acid molecule of being identified;

(d) methionine level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give methionine level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that methionine(Met) increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control methionine level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

[0554.0.0.0] summary

The present invention relates in microorganism, vegetable cell, plant, plant tissue or its one or more parts, produce the method for fine chemicals.The present invention also relates to nucleic acid molecule, polypeptide, nucleic acid construct, carrier, antisense molecule, antibody, host cell, plant tissue, reproductive material, results material, plant, microorganism and Pestcidal compositions and their purposes in addition.

Produce the method for fine chemicals

[0000.0.0.1] the present invention relates to produce the another kind of method of following defined fine chemicals and corresponding embodiment as described below in another embodiment.

[0001.0.0.1] to [0007.0.0.1]: see that [0001.0.0.0] is to [0007.0.0.0]

The certain enzyme in the amino acid biosynthetic pathway is gone to regulate [0007.1.0.1] and the method that improves the free threonine level is disclosed in United States Patent (USP) 5,942,660, this can express realization by crossing of E.C. 2.7.2.4., homoserine dehydrogenase or threonine synthase wild-type or that go to regulate.

[0008.0.0.1] sees [0008.0.0.0]

[0009.0.1.1] as mentioned above, indispensable amino acid is that people and many Mammalss such as domestic animal are necessary.Threonine be important moiety and Threonine for the formation of enamel, collagen and elastin is essential in many body proteins, and wherein collagen and elastin are that healthy skin and wound healing is needed.Threonine is the precursor of glycine and Serine.The fat that Threonine is being controlled in the liver as lipotropic agent accumulates.Threonine is an immunologic stimulant, because it promotes thymus gland growth and active.Threonine is digestive ferment and intestine immunity secretory product, particularly mucinous composition.Threonine has been used as additive so that help to alleviate anxiety and some depressed situations.In animal produces, Threonine is as important indispensable amino acid, for for the pig normally second limiting amino acid and for chicken (jungle fowl (Gallus gallus f.domestica), for example laying hen or meat chicken) be generally the 3rd limiting amino acid.

[0010.0.0.1] sees [0010.0.0.0]

[0011.0.0.1] sees [0011.0.0.0]

[0012.0.1.1] the objective of the invention is to develop the method for the economy of synthetic Threonine, preferred L-Threonine.The same with Methionin and methionine(Met), Threonine is one of the most normal amino acid that is restricted (depending on biology).

[0013.0.0.1] sees [0013.0.0.0]

[0014.0.1.1] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is Threonine, preferred L-Threonine.Therefore, in the present invention, term " fine chemicals " is meant " Threonine " as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of Threonine as used herein.

[0015.0.1.1] an embodiment, term " fine chemicals " meaning is meant Threonine, preferred L-Threonine.In whole specification sheets, term " fine chemicals " meaning is meant salt, ester or the acid amides of Threonine, preferred L-Threonine, its free form or is bonded to proteinic Threonine, preferred L-Threonine.In preferred embodiments, term " fine chemicals " meaning is meant Threonine, preferred L-Threonine or its salt of free form or is bonded to proteinic Threonine, preferred L-Threonine.

[0016.0.1.1] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YFL050C, YKR057W, YIL150C, YNL046W, YNL120C, b0186, b0730, b1829, b2170, b0019, b0464, b1360, b1738, b1830, b1896, b2270, b2414, b2552, b2664, b3074, b3160, b3231, b3462, b3791, b3966, b4004, YOR245C; With

(b) in allowing described biology, produce under the condition that fine chemicals is Threonine or the fine chemicals that comprises Threonine and make biological growth.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II A or IIB the 3rd row, 6-15,339-355 capable shown in activity of proteins, perhaps have by Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) under the condition that allows generation fine chemicals, particularly Threonine, make biological growth.

[0017.0.0.1] sees [0017.0.0.0]

[0018.0.0.1] sees [0018.0.0.0]

The method that [0019.0.1.1] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare the fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II A or IIB the 3rd row, 6-15, protein active shown in 339-355 is capable or by as Table II A or IIB the 5th or 7 row, 6-15, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 339-355 was capable.

[0020.0.1.1] be surprisingly found out that, at least a as Table II A or IIB the 3rd row, 6-10 is capable and the 355th row shown in yeast saccharomyces cerevisiae protein and/or at least aly give Threonine (or fine chemicals) the content raising of institute conversion plant as the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II A or IIB the 3rd row, 11-15,339-354 are capable.

[0021.0.0.1] sees [0021.0.0.0]

The sequence of [0022.0.1.1] yeast saccharomyces cerevisiae YFL050C has been published in Murakami etc., Nat.Genet.10 (3), 261-268,1995 and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be two-trivalent inorganic cation transporter.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product that is defined as two-trivalent inorganic cation transporter of yeast saccharomyces cerevisiae or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the L-Threonine of free or combining form.

The sequence of yeast saccharomyces cerevisiae YKR057W has been published in Dujon etc., Nature 369 (6479), 371-378,1994 and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be to participate in that rrna is biological to be taken place and ribosomal protein translation, that be similar to the S21 ribosomal protein.Therefore, in one embodiment, the inventive method comprises the biological generation of the participation rrna from yeast saccharomyces cerevisiae as shown here and the purposes of ribosomal protein or its homologue translation, that be similar to the S21 ribosomal protein, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the L-Threonine of free or combining form.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Goffeau etc., Science 274 (5287), 546-547,1996 and Churcher etc., Nature 387 (6632 supplementary issue), 84-87,1997, and its activity to be defined as be that the S phase (DNA synthetic) is initial or finish necessary chromobindins.Therefore, in one embodiment, the inventive method comprises that the S phase from yeast saccharomyces cerevisiae as shown here (DNA synthetic) is initial or finishes the purposes of necessary chromobindins or its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the L-Threonine of free or combining form.

The sequence of yeast saccharomyces cerevisiae YNL046W has been published in Goffeau etc., and Science 274 (5287), 546-547,1996 and Philippsen etc., Nature 387 (6632 supplementary issue), 93-98,1997, and its activity to be defined as be possible endoplasmic reticulum albumen.Therefore, in one embodiment, the inventive method comprises the proteic YNL046W of endoplasmic reticulum that the conduct from yeast saccharomyces cerevisiae as shown here is possible or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.

The sequence of yeast saccharomyces cerevisiae YNL120C has been published in de Antoni etc., Yeast 13:261-266, and 1997, and its cytoactive is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YNL120C from yeast saccharomyces cerevisiae as shown here, being used for as described producing fine chemicals in biological or its part is Threonine, particularly increases Threonine, the preferred quantity of the Threonine of free or combining form.

The sequence of e. coli k12 b0186 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be lysine decarboxylase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary lysine decarboxylase or its homologue purposes, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the lysine decarboxylase activity is enhanced or produces in the methods of the invention, and for example the activity from colibacillary lysine decarboxylase or its homologue is enhanced or produces.

The sequence of e. coli k12 b0730 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be that succinic thiokinase operon transcriptional and acyl are replied regulatory gene.Therefore, in one embodiment, the inventive method comprises purposes of replying regulatory gene or its homologue from colibacillary succinic thiokinase operon transcriptional or lipid acid as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the L-Threonine of free or combining form.

The sequence of e. coli k12 b1829 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be heat shock protein.Therefore, in one embodiment, the inventive method comprises the purposes from intestinal bacteria " heat shock protein " or its homologue as shown here, it is used for to be Threonine, particularly to increase the quantity of the Threonine of free or combining form at biological or its part generation fine chemicals as described.In one embodiment, the activity of htpX heat shock protein is enhanced or produces in the methods of the invention, and for example the activity from colibacillary htpX heat shock protein or its homologue is enhanced or produces.The htpX heat shock protein also is noted as has protease activity.Therefore, in one embodiment, in the methods of the invention proteolytic enzyme, preferred heat shock protein(HSP) enzyme, more preferably the activity of htpX proteolytic enzyme or its homologue is enhanced, being used for as shown producing fine chemicals in biological or its part is Threonine, particularly increases the quantity of the Threonine of free or combining form.

The sequence of e. coli k12 b2170 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be sugar outflow transporter.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary sugar outflow transporter B or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the L-Threonine of free or combining form.

The sequence of e. coli k12 b0019 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the protein that is used to transport; The protein of transhipment small molecules, preferred cationic.In a more preferred embodiment, protein have to coerce, the Na+/H+ antiport protein-active of high salt and pH reaction particularly.Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary protein that is used to transport, the preferred proteic purposes of stress reaction Na+/H+ antiport, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the L-Threonine of free or combining form.

The sequence of e. coli k12 b0464 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the transcription repressor (TetR/AcrR family) of multiple medicines outflow pump.Therefore, in one embodiment, the inventive method comprises as shown here from the transcription repressor (TetR/AcrR family) of colibacillary multiple medicines outflow pump or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity of the transcription repressor (TetR/AcrR family) of the pump of multiple medicines outflow in the methods of the invention is enhanced or produces, and for example is enhanced or produces from the transcription repressor (TetR/AcrR family) of intestinal bacteria multiple medicines outflow pump or the activity of its homologue.

The sequence of e. coli k12 b1360 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition dna replication protein matter.Therefore, in one embodiment, the inventive method comprises as shown here from the dna replication protein matter of colibacillary supposition or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity of Jia Ding dna replication protein matter is enhanced or produces in the methods of the invention, for example is enhanced or produces from the dna replication protein matter of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b1738 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be PEP dependency phosphotransferase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary PEP dependency phosphotransferase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, PEP dependency phosphate transferase activity is enhanced or produces in the methods of the invention, and for example the activity from colibacillary PEP dependency phosphotransferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b1830 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the carboxyl terminal proteolytic enzyme of penicillin-binding protein 4.Therefore, in one embodiment, the inventive method comprises as shown here from the carboxyl terminal proteolytic enzyme of colibacillary penicillin-binding protein 4 or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the carboxyl terminal protease activity of penicillin-binding protein 4 is enhanced or produces in the methods of the invention, and for example carboxyl terminal proteolytic enzyme or its homologue activity from colibacillary penicillin-binding protein 4 is enhanced or produces.

The sequence of e. coli k12 b1896 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be trehalose-6-phosphate synthase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary trehalose-6-phosphate synthase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the trehalose-6-phosphate synthase activity is enhanced or produces in the methods of the invention, and for example the activity from colibacillary trehalose-6-phosphate synthase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2414 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit activity are enhanced or produce in the methods of the invention, and for example the activity from colibacillary PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue is enhanced or produces.

The sequence of e. coli k12 b2552 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be dihydropteridine reductase (nitric oxide dioxygenase).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary dihydropteridine reductase (nitric oxide dioxygenase) or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, dihydropteridine reductase (nitric oxide dioxygenase) activity is enhanced or produces in the methods of the invention, for example is enhanced or produces from colibacillary dihydropteridine reductase (nitric oxide dioxygenase) or its homologue activity.

The sequence of e. coli k12 b4004 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be transcription regulatory protein.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary transcription regulatory protein or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the transcription regulatory protein activity is enhanced or produces in the methods of the invention, and for example the activity from colibacillary transcription regulatory protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b2664 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be supposition have the transcription repressor (GntR family) of DNA in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprises as shown here having DNA in conjunction with the transcription repressor (GntR family) in wing spirane structure territory or the purposes of its homologue from colibacillary supposition, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the DNA that has of supposition in the methods of the invention is enhanced or produces in conjunction with the activity of the transcription repressor (GntR family) in wing spirane structure territory, and for example the DNA that has from colibacillary supposition is enhanced or produces in conjunction with the transcription repressor (GntR family) in wing spirane structure territory or the activity of its homologue.

The sequence of e. coli k12 b 3074 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises as shown here from the tRNA synthetic enzyme of colibacillary supposition or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity of Jia Ding tRNA synthetic enzyme is enhanced or produces in the methods of the invention, for example is enhanced or produces from the tRNA synthetic enzyme of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b2270 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary protein b2270 or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, be enhanced or produce by the coded activity of proteins of b2270 in the methods of the invention, for example be enhanced or produce from colibacillary activity by the coded protein of b2270 or its homologue.

The sequence of e. coli k12 b3160 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the monooxygenase with luciferase sample atpase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the monooxygenase with luciferase sample atpase activity of colibacillary supposition or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity of Jia Ding the monooxygenase with luciferase sample atpase activity is enhanced or produces in the methods of the invention, for example is enhanced or produces from the monooxygenase with luciferase sample atpase activity of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b3231 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L13.Therefore, in one embodiment, the inventive method comprises the purposes from intestinal bacteria 50S ribosomal subunit protein matter L13 or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity of 50S ribosomal subunit protein matter L13 is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 50S ribosomal subunit protein matter L13 or its homologue is enhanced or produces.

The sequence of e. coli k12 b3462 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the conformity membrane cell division protein.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary conformity membrane cell division protein or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity of conformity membrane cell division protein is enhanced or produces in the methods of the invention, and for example the activity from colibacillary conformity membrane cell division protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b3791 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be to participate in the biosynthetic transaminase of lipopolysaccharides.Therefore, in one embodiment, the inventive method comprises the purposes from the intestinal bacteria participation biosynthetic transaminase of lipopolysaccharides or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity that participates in the biosynthetic transaminase of lipopolysaccharides in the methods of the invention is enhanced or produces, and for example the activity from the biosynthetic transaminase of colibacillary participation lipopolysaccharides or its homologue is enhanced or produces.

The sequence of e. coli k12 b3966 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be outer porin.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary outer porin or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the Threonine of free or combining form.In one embodiment, the activity of outer porin is enhanced or produces in the methods of the invention, and for example the activity from colibacillary outer porin or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YOR245C has been published in Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and its activity to be defined as be acetyl-CoA: Diacrylglycerol acyl transferase.Therefore; in one embodiment; the inventive method comprises the acetyl-CoA that is defined as from yeast saccharomyces cerevisiae as shown here: the gene product of Diacrylglycerol acyl transferase or the purposes of its homologue; as described its to be used for producing fine chemicals in biological or its part be Threonine, particularly increase Threonine, the preferred quantity of the L-Threonine of free or combining form.In one embodiment, acetyl-CoA in the methods of the invention: the activity of Diacrylglycerol acyl transferase is enhanced or produces, and for example from the acetyl-CoA of yeast saccharomyces cerevisiae: the activity of Diacrylglycerol acyl transferase or its homologue is enhanced or produces.

The homologous compound (=homologue) of [0023.0.1.1] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with protein shown at least a Table II A or IIB the 3rd row, 6-15,339-355 are capable, for example have by contain Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.In one embodiment, shown in Table II A or IIB 6-10,339-355 are capable in the polypeptide homologue of any one be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in the biology, and described homologue is from eukaryote.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 11-15,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from bacterium.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 6-10,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from fungi.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 11-15,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Proteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 6-10,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Ascomycota.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 11-15,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 6-10,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from yeast.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 11-15,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the enterobacteria order.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB, the 3rd row, 6-15,339-355 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 11-15,339-354 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from enterobacteriaceae.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II A or IIB the 3rd row, 6-10,355 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetes.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II A or IIB the 3rd row, 11-15,339-354 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Escherichia.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II A or IIB the 3rd row, 6-10,355 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetaceae.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II A or IIB the 3rd row, 6-10,355 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.

[0023.1.0.1] as the homologue of the polypeptide shown in Table II A or IIB, the 3rd row, 6-15,339-355 are capable can be by Table I A or IB the 7th row, 6-10,339-355 capable shown in nucleic acid molecule encoding polypeptide or can be Table II A or IIB the 7th row, 6-10,339-355 capable shown in polypeptide.As the homologue of polypeptide shown in Table II A or IIB, the 3rd row, 6-15,339-355 are capable can be by Table I A or IB the 7th row, 6-10,339-355 capable shown in nucleic acid molecule encoding polypeptide or can be Table II A or IIB the 7th row, 11-15,339-355 capable shown in polypeptide.

[0024.0.0.1]: see [0024.0.0.0]

[0025.0.1.1] is according to the present invention, the Threonine level increases in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II A or IIB, the 3rd row, 6-15,339-355 capable shown in activity of proteins.In this manual, if this kind protein or polypeptide still have Table II A or IIB, the 3rd row, 6-15, any one proteinic biology or the enzyme activity shown in 339-355 is capable, if promptly with Table II A or IIB the 3rd row, 6-10, any one protein shown in 339-355 is capable is compared and/or is listed as with Table II A or IIB the 3rd, 11-15, any one protein was compared shown in 339-354 was capable, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.1] in one embodiment, if it comes comfortable the evolution to go up biology with the biological apart from each other of expressing it, polypeptide of the present invention is still given described activity, as increase fine chemicals in biological or its part.For example play eozoan and express biology from different sections, order, guiding principle or door.

[0026.0.0.1] to [0033.0.0.1]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.1.1] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, its have Table II A or IIB, the 3rd row, 6-15,339-355 capable shown in protein or by Table I A or IB the 5th row, 6-15,339-355 capable shown in the protein of nucleic acid molecule encoding or its homologue (as homologue shown in Table I A or IB the 7th row, 6-15,339-355 are capable) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows the fine chemicals quantity that increases.

[0035.0.0.1] to [0044.0.0.1]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.1.1] in one embodiment, under the situation that yeast saccharomyces cerevisiae protein YFL050C or two-trivalent inorganic cation transporter or its homologue (shown in Table I A or IB the 5th or 7 row, the 6th row) activity is enhanced, preferably, giving the fine chemicals Threonine is increased between 19% and 56%.

Yeast saccharomyces cerevisiae protein YKR057W or participate in rrna biological take place and situation that ribosomal protein translation, that be similar to the S21 ribosomal protein or its homologue (shown in Table I A or IB the 5th or 7 row, the 7th row) activity is enhanced under, preferably, giving the fine chemicals Threonine in one embodiment is increased between 34% and 142%.

In one embodiment, initial or finish necessary chromobindins or its homologue in yeast saccharomyces cerevisiae protein YIL150C or " protein s phase (DNA synthetic) initial or finish institute essential " or S phase (DNA synthesizes), under the situation that cell division cycle protein matter (for example shown in Table I A or IB the 5th or 7 row, the eighth row) activity is enhanced, preferably, giving the fine chemicals Threonine is increased between 25% and 319%.

At yeast saccharomyces cerevisiae protein YNL046W or its homologue, under the situation that for example possible endoplasmic reticulum albumen (shown in Table I A or IB the 5th or 7 row, the 9th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 18% and 53%.

In one embodiment, under the situation that yeast saccharomyces cerevisiae protein YNL120C or its homologue (shown in Table I A or IB the 5th or 7 row, the 10th row) activity is enhanced, preferably, giving the fine chemicals Threonine increases by 44%.

In one embodiment, under the situation that e. coli k12 protein b0186 or lysine decarboxylase or its homologue (shown in Table I A or IB the 5th or 7 row, the 11st row) activity is enhanced, preferably, giving the fine chemicals Threonine is increased between 49% and 228%.

In one embodiment, at e. coli k12 protein b0730 or have and be defined as under the situation that the active protein of succinic thiokinase operon transcriptional or its homologue (shown in Table I A or IB the 5th or 7 row, the 12nd row) activity is enhanced, preferably, giving the fine chemicals Threonine is increased between 53% and 177%.

At e. coli k12 protein b1829 or under the situation that its homologue activity is enhanced, for example protease activity is enhanced, preferably the heat shock protein activity is enhanced, more preferably htpX protein or its homologue (shown in Table I A or IB the 5th or 7 row, the 13rd row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 17% and 114%.

Under the situation that e. coli k12 protein b2170 or sugar outflow transporter or its homologue (shown in Table I A or IB the 5th or 7 row, the 14th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 35% and 79%.

At e. coli k12 protein b0019 or transport cationic protein or its homologue, for example as Na ⁺/ H ⁺Under the situation that antiport albumen (shown in Table I A or IB the 5th or 7 row, the 15th row) activity is enhanced, preferably, give the fine chemicals Threonine in one embodiment and be increased between 24% and 44%.

In one embodiment, at e. coli k12 protein b0464 or have under the situation that transcription repressor (TetR/AcrR family) the active protein that is defined as multiple medicines outflow pump or its homologue (shown in Table I A or IB the 5th or 7 row, the 339th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 23% and 43%.

In one embodiment, e. coli k12 protein b1360 have the active protein of dna replication protein matter of the supposition of being defined as or situation that its homologue (shown in Table I A or IB the 5th or 7 row, the 340th row) activity is enhanced under, preferably, giving the fine chemicals Threonine in one embodiment is increased between 16% and 38%.

In one embodiment, at e. coli k12 protein b1738 or have under the situation that the protein that is defined as PEP dependency phosphate transferase activity or its homologue (shown in Table I A or IB the 5th or 7 row, the 341st row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 27% and 361%.

In one embodiment, at e. coli k12 protein b1830 or have under the situation that the protein of the carboxyl terminal protease activity that is defined as penicillin-binding protein 4 or its homologue (shown in Table I A or IB the 5th or 7 row, the 342nd row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 24% and 43%.

In one embodiment, at e. coli k12 protein b1896 or have and be defined as under the situation that the active protein of trehalose-6-phosphate synthase or its homologue (shown in Table I A or IB the 5th or 7 row, the 343rd row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 46% and 108%.

In one embodiment, at e. coli k12 protein b2414 or have and be defined as under the situation that PLP dependent enzyme cysteine synthase A and the active protein of O-acetylserine sulfhydrylase A subunit or its homologue (shown in Table I A or IB the 5th or 7 row, the 345th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 24% and 46%.

In one embodiment, at e. coli k12 protein b2552 or have under the situation that the active protein that is defined as dihydropteridine reductase (nitric oxide dioxygenase) or its homologue (shown in Table I A or IB the 5th or 7 row, the 346th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 17% and 37%.

In one embodiment, at e. coli k12 protein b4004 or have under the situation that the active protein that is defined as transcription regulatory protein or its homologue (shown in Table I A or IB the 5th or 7 row, the 354th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 17% and 37%.

In one embodiment, at e. coli k12 protein b2664 or have the having under the situation that DNA is enhanced in conjunction with the active protein of the transcription repressor (GntR family) in wing spirane structure territory or its homologue (shown in Table I A or IB the 5th or 7 row, the 347th row) activity of the supposition of being defined as, preferably, giving the fine chemicals Threonine in one embodiment is increased between 29% and 284%.

In one embodiment, e. coli k12 protein b3074 have the active protein of tRNA synthetic enzyme of the supposition of being defined as or situation that its homologue (shown in Table I A or IB the 5th or 7 row, the 348th row) activity is enhanced under, preferably, giving the fine chemicals Threonine in one embodiment is increased between 31% and 59%.

In one embodiment, under the situation that e. coli k12 protein b2270 or its homologue (shown in Table I A or IB the 5th or 7 row, the 344th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 31% and 59%.

In one embodiment, e. coli k12 protein b3160 have the active protein of monooxygenase of the supposition of being defined as with luciferase sample atpase activity or situation that its homologue (shown in Table I A or IB the 5th or 7 row, the 349th row) activity is enhanced under, preferably, giving the fine chemicals Threonine in one embodiment is increased between 25% and 56%.

In one embodiment, at e. coli k12 protein b3231 or have under the situation that the active protein that is defined as 50S ribosomal subunit protein matter L13 or its homologue (shown in Table I A or IB the 5th or 7 row, the 350th row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 17% and 32%.

In one embodiment, at e. coli k12 protein b3462 or have under the situation that the active protein that is defined as the conformity membrane cell division protein or its homologue (shown in Table I A or IB the 5th or 7 row, the 351st row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 18% and 51%.

In one embodiment, at e. coli k12 protein b3791 or have and be defined as under the situation that the active protein that participates in the biosynthetic transaminase of lipopolysaccharides or its homologue (shown in Table I A or IB the 5th or 7 row, the 352nd row) activity is enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 38% and 44%.

In one embodiment, at e. coli k12 protein b3966 or have be defined as outside under the active protein of porin or its homologue (shown in Table I A or IB the 5th or 7 row, the 353rd row) the activity situation about being enhanced, preferably, giving the fine chemicals Threonine in one embodiment is increased between 19% and 47%.

At yeast saccharomyces cerevisiae protein YOR245C or have and be defined as acetyl-CoA: under the situation that the active protein of Diacrylglycerol acyl transferase or its homologue (shown in Table I A or IB the 5th or 7 row, the 355th row) activity is enhanced; preferably, giving the fine chemicals Threonine in one embodiment is increased between 18% and 81%.

[0046.0.1.1] under the situation that yeast saccharomyces cerevisiae protein YFL050C or its homologue (as two-trivalent inorganic cation transporter) activity are enhanced, preferably, gives between fine chemicals Threonine and the L-Ala and increasing in one embodiment.

In one embodiment, at yeast saccharomyces cerevisiae protein YKR057W or its homologue, under the situation that the activity that for example participates in the biological generation of rrna and translate, be similar to the ribosomal protein of S21 ribosomal protein is enhanced, preferably, giving fine chemicals Threonine and arginine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example under the situation that the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue is enhanced, preferably, giving fine chemicals Threonine and fumaric acid increases.

At e. coli k12 protein b0186 or its homologue, for example under the situation that the activity of lysine decarboxylase or its homologue is enhanced, preferably, giving fine chemicals Threonine and methionine(Met) increases.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example have and be defined as succinic thiokinase operon transcriptional and acyl and reply under the situation that the activity of the active protein of regulatory gene or its homologue is enhanced, preferably, giving fine chemicals Threonine and β-Hu Luobusu increases.

In one embodiment, activity at e. coli k12 protein b1829 or its homologue is enhanced, for example protease activity is enhanced, preferably the heat shock protein activity is enhanced, more preferably under the situation that htpX protein or its homologue activity are enhanced, preferably, giving fine chemicals Threonine and C18:0 increases.

In one embodiment, be enhanced at e. coli k12 protein b2170 or its homologue activity, for example under the situation that sugar outflow transporter B activity is enhanced, preferably, giving fine chemicals Threonine and isopentenylpyrophosphate increases.

In one embodiment, at e. coli k12 protein b0019 or its homologue, for example transport cationic protein or its homologue, under the situation about being enhanced as the proteic activity of Na+/H+ antiport, preferably, giving fine chemicals Threonine and β-Gu Zaichun increases.

[0047.0.0.1] sees [0047.0.0.0]

[0048.0.0.1] sees [0048.0.0.0]

[0049.0.1.1] has to give and improves fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, 6-15, the polypeptide of consensus sequence shown in 339-355 is capable, perhaps as Table II A or IIB the 5th or 7 row, 6-15, its function homologue shown in 339-355 is capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, 6-15, nucleic acid molecule shown in 339-355 is capable or its function homologue as herein described) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.1.1] for the purposes of the present invention, term " Threonine " and " L-Threonine " also comprise corresponding salt, for example Threonine hydrochloride or Threonine vitriol.Preferably, the term Threonine is intended to comprise term L-Threonine.

[0051.0.0.1] sees [0051.0.0.0]

[0052.0.0.1] sees [0052.0.0.0]

[0053.0.1.1] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention (for example having as the active polypeptide of protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in)) and is expressed and increase, and has the activity of the raising fine chemicals described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention (for example have as protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II the 5th or 7 row, 1-5 is capable or 334-338 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of the raising Threonine described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give the raising Threonine that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in), perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give the raising Threonine that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give the raising Threonine that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in);

(f) express the transgenosis of coded protein, described protein give the raising Threonine that has described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have the raising Threonine described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide (for example having as the active polypeptide of protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in)).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) (heat shock protein(HSP) for example of the present invention) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.1.1] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example give Threonine after the active expression of polypeptides of protein or its homologue (for example Table II A or IIB the 7th row, 6-15,339-355 capable shown in polypeptide) or the activity shown in capable and increase improving coded protein or have as Table II the 5th row, 6-15,339-355.

[0055.0.0.1] to [0064.0.0.1] sees that [0055.0.0.0] is to [0064.0.0.0]

The activation (for example giving fine chemicals after improving expression or activity increases) that [0065.0.1.1] also can improve the activation with above-mentioned active endogenous polypeptide, polypeptide of the present invention by introducing synthetic transcription factor, the coding region of described synthetic transcription factor and endogenous polypeptide of the present invention or its endogenous homologue encoding gene are combined closely and are activated it and transcribe.Can resolve the chimeric zinc finger protein that contains specific DNA binding domains and activation structure territory (for example VP16 structural domain of hsv).Specific binding domains can be in conjunction with the regulatory region of endogenous protein coding region.The specifically expressing of the chimeric transcription factor expression in biological (particularly plant) causing endogenous polypeptide of the present invention (particularly its plant homologue), see for example WO01/52620, Oriz, Proc.Natl.Acad.Sci.USA, 2002, the 99 volumes, 13290 or Guan, Proc.Natl.Acad.Sci.USA, 2002, the 99 volumes, 13296.

[0066.0.0.1] to [0069.0.0.1]: see that [0066.0.0.0] is to [0069.0.0.0]

[0070.0.1.1] imports biology separately or with other assortment of genes owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, the favorable amino acid composition that for example contains the fine chemicals of high level (from the physiology of nutrition angle) particularly is that amino acid equally also is fine chemicals.

[0071.0.0.1] sees [0071.0.0.0]

[0072.0.1.1] therefore might produce more favourable hydroxy-containing compounds in the methods of the invention by influencing metabolism.The example of this compounds also has Serine, homoserine, phosphohomoserine or oxyproline or methionine(Met) except Threonine.

[0073.0.1.1] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of polypeptide of the present invention or its homologue (for example Table II A or IIB the 5th or 7 row, 6-15,339-355 capable shown in) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, the fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or bonded fine chemicals by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation, and optional other dissociates and/or in conjunction with amino acid.

[0074.0.0.1] to [0084.0.0.1]: see that [0074.0.0.0] is to [0084.0.0.0]

[0085.0.1.1] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable, promotor for example, perhaps (c) (a) with (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.1]: see [0086.0.0.0]

[0087.0.0.1]: see [0087.0.0.0]

[0088.0.1.1] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified aminoacids content.Often be subject to minority indispensable amino acid (as Methionin, Threonine or methionine(Met)) owing to for example be used to raise the nutritive value of the plant of monogastric animal, so this is very important for the plant breeder.

[0088.1.1.1] in one embodiment, after improving or having produced the activity of polypeptide of the present invention, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, the transgenic plant that produced can grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0089.0.0.1] to [0097.0.0.1]: see that [0089.0.0.0] is to [0097.0.0.0]

[0098.0.1.1] in another embodiment, the fine chemicals Threonine be produce according to the present invention and carry out isolating where necessary.It is favourable as methionine(Met), Methionin and/or aminoacid mixture that the method according to this invention produces other amino acid.

[0099.0.0.1] to [0102.0.0.1]: see that [0099.0.0.0] is to [0102.0.0.0]

[0103.0.1.1] in preferred embodiments, the present invention relates to produce the method for fine chemicals Threonine, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) the preferred nucleic acid molecule of encoding mature form polypeptide at least, described polypeptide have as the sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as the sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals Threonine quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals Threonine quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals Threonine quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals Threonine quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals Threonine quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer as sequence shown in Table III the 7th row, 6-15,339-355 are capable obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of fine chemicals Threonine quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals Threonine quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as the consensus sequence of sequence shown in Table IV the 7th row, 6-15,339-355 are capable and give biology or its part in the nucleic acid molecule that increases of fine chemicals Threonine quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of fine chemicals Threonine quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding schedule IIA or IIB the 5th or 7 row, 6-15,339-355 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals Threonine quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0104.0.1.1] in one embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention not by Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in sequence form.In another embodiment, nucleic acid molecule not coding schedule IA or IB the 5th or 7 row, 6-15,339-355 capable shown in polypeptide of sequence.

[0105.0.0.1] to [0107.0.0.1]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.1.1] method of the present invention and had as Table I A or IB the 5th or 7 row, 6-15, the nucleic acid molecule of sequence shown in 339-355 is capable, from being listed as Table II A or IIB the 5th or 7,6-15, aminoacid sequence shown in 339-355 is capable is derived or is listed as from containing just like Table IV the 7th, 6-15, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 339-355 is capable, perhaps its coding has as Table II A or IIB the 3rd, 5 or 7 row, 6-15, active or the bioactive polypeptide of polypeptidase shown in 339-355 is capable or for example give derivative or the homologue that the fine chemicals Threonine increases behind its expression or active the increasing.

[0109.0.0.1] sees [0109.0.0.0]

[0110.0.1.1] helps the nucleic acid molecule that the inventive method and coding have the polypeptide of polypeptide active of the present invention and can determine from generally open database.

[0111.0.0.1] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.1.1] the inventive method is the isolated nucleic acid sequences form, its coding has as the polypeptide of polypeptide active shown in Table II A or IIB, the 3rd row, 6-15,339-355 are capable or has polypeptide as peptide sequence shown in Table II A or IIB the 5th and 7 row, 6-15,339-355 are capable, and gives the fine chemicals Threonine and increase.

[0113.0.0.1] to [0120.0.0.1]: see that [0113.0.0.0] is to [0120.0.0.0]

[0121.0.1.1] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with the difference that one or more amino acid moleculars are arranged as peptide sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or its functional homologue as herein described, described artificial sequence is preferably given aforementioned activity, and promptly giving the fine chemicals Threonine after improving its activity increases.

[0122.0.0.1] to [127.0.0.1]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.1.1] polymerase chain reaction (PCR) amplification is used (for example as the primer shown in Table III the 7th row, 6-15,339-355 are capable to) can be based on as the sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable or from producing as sequence deutero-sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.

[0129.0.1.1] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of the inventive method (the particularly sequence of polypeptide of the present invention).Indicate in the shown in the drawings comparison of the conservative region of polypeptide of the present invention.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence was from described comparison shown in Table IV the 7th row, 6-15,339-355 were capable.

[0130.0.1.1] can use degenerated primer to have the new protein fragment of aforementioned activity (increasing as give fine chemicals after improving expression or activity) by pcr amplification then or derive from other functional homologue of the polypeptide of the present invention of other biological.

[0131.0.0.1] to [0138.0.0.1]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.1.1] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving Threonine increases), described dna sequence dna under loose hybridization conditions with Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in sequence hybridization, and coding is expressed and is had the active peptide of the Threonine of increasing.

[0140.0.0.1] to [0146.0.0.1]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.1.1] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.1.1] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I A or IB the 5th or 7 row, 6-15, nucleotide sequence shown in 339-355 is capable or its funtion part homology are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases fine chemicals.

[0149.0.1.1] nucleic acid molecule of the present invention comprises nucleotide sequence, one of nucleotide sequence or the hybridization of its part shown in described sequence and Table I A or IB the 5th or 7 row, 6-15,339-355 are capable, preferably in as literary composition, hybridize under the defined stringent condition, and coding has the protein of above-mentioned activity (as giving the fine chemicals increase).

[00149.1.1.1] randomly, with Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity is for being known activity or being used for these protein of note as the protein shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable.

[0150.0.1.1] in addition, nucleic acid molecule of the present invention can only contain Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving the fine chemicals Threonine when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in sense strand, Table I A or the IB the 5th of one of sequence or 7 row, 6-15,339-355 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 6-15,339-355 capable shown in the right PCR of primer will produce as Table I A or IB the 5th or 7 be listed as, 6-15,339-355 capable shown in the fragment of polynucleotide sequence.

[0151.0.0.1] sees [0151.0.0.0]

[0152.0.1.1] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as the abundant homology of aminoacid sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable, make this albumen or its part keep to participate in produce the ability of Threonine, specifically be included in plant or the microorganism as described above or the activity of raising Threonine as be shown in the examples.

[0153.0.1.1] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing Threonine.In one embodiment, as protein shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or its part have Table II A for example or IIB the 3rd row, 6-15,339-355 capable shown in the activity of polypeptide.

[0154.0.1.1] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving fine chemicals increases.

[0155.0.0.1] sees [0155.0.0.0]

[0156.0.0.1] sees [0156.0.0.0]

[0157.0.1.1] the present invention relate in addition owing to the genetic code degeneracy be different from Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology Threonine increase) for example comprises as the polypeptide of consensus sequence shown in Table IV the 5th or 7 row, 6-15,339-355 are capable or as the nucleic acid molecule of polypeptide shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or its function homologue.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as Table IV the 5th or 7 row, 6-15, the 339-355 consensus sequence shown in capable or as polypeptide or its function homologue shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as the consensus sequence shown in Table IV the 7th row, 6-15,339-355 are capable or as the basic homology of aminoacid sequence of polypeptide shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I A the 5th or 7 row, 6-15, the 339-355 sequence shown in capable.

[0158.0.0.1] to [0160.0.0.1]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.1.1] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.1]: see [0162.0.0.0]

[0163.0.1.1] preferably, under stringent condition with Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.1]: see [0164.0.0.0]

[0165.0.1.1] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in sequence).

[0166.0.0.1]: see [0166.0.0.0]

[0167.0.0.1] sees [0167.0.0.0]

[0168.0.1.1] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are with different as the contained sequence of sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable, but have kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the aminoacid sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II A or IIB the 5th or 7 be listed as, 6-15, sequence shown in 339-355 is capable is identical at least about 60%, more preferably with as Table II A or IIB the 5th or 7 be listed as, 6-15, one of sequence shown in 339-355 is capable is identical at least about 70%, even more preferably with as Table II A or IIB the 5th or 7 be listed as, 6-15, sequence shown in 339-355 is capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II A or IIB the 5th or 7 be listed as, 6-15, sequence shown in 339-355 is capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.1] to [0172.0.0.1]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.1.1] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:40199 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:40199 sequence before use.

[0174.0.0.1]: see [0174.0.0.0]

[0175.0.1.1] for example, the sequence that has 80% homology at protein level and SEQ ID NO:40200 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:40200 sequence.

[0176.0.1.1] is by replacing, insert or lack from being listed as Table II A or IIB the 5th or 7 according to of the present invention, 6-15, the functional equivalent that one of polypeptide obtained shown in 339-355 was capable be listed as according to of the present invention as Table II A or IIB the 5th or 7,6-15, one of polypeptide had at least 30% shown in 339-355 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II A or IIB the 5th or 7 row, 6-15, polypeptide shown in 339-355 is capable has essentially identical character and discerns.

[0177.0.1.1] is by replacing, insert or lack from being listed as Table I A or IB the 5th or 7 according to of the present invention, 6-15, the functional equivalent that nucleotide sequence obtained shown in 339-355 was capable be listed as according to of the present invention as Table II A or IIB the 5th or 7,6-15, one of polypeptide had at least 30% shown in 339-355 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II A or IIB the 5th or 7 be listed as, 6-15, the polypeptide of the essentially identical character of polypeptide shown in 339-355 is capable.

[0178.0.0.1] sees [0178.0.0.0]

[0179.0.1.1] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in), and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II A or IIB the 5th or 7 be listed as, 6-15,339-355 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through to introduce sudden change in the encoding sequence of standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) sequence shown in capable as Table I A or IB the 5th or 7 row, 6-15,339-355.

[0180.0.0.1] to [0183.0.0.1]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.1.1] is employed to have as Table I A or IB the 5th or 7 row, 6-15, the nucleotide sequence homologous compound of sequence shown in 339-355 is capable, perhaps come Table II A or IIB the 5th or 7 row freely, 6-15, the homologous compound of the nucleotide sequence of sequence shown in 339-355 is capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.1.1] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as the sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable.In one embodiment, the preferred nucleic acid molecule comprises other nucleotide sequence that does not show in the least possible, the arbitrary sequence shown in capable as Table I A or IB the 5th or 7 row, 6-15,339-355.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method is with identical as the sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable.

The employed one or more nucleic acid molecule encodings of [0186.0.1.1] also preferred the inventive method comprise as polypeptide of sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the coded polynucleotide that are used for the inventive method are with identical as sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.

[0187.0.1.1] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as polypeptide of sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable and contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with identical as the encoding sequence of sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.

The polypeptide (=protein) that [0188.0.1.1] still has the polypeptide basic enzyme activity of the present invention (being its active basic reduction that do not have) of giving fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II A or IIB the 5th or 7 be listed as, 6-15, polypeptide active shown in 339-355 is capable, preferably with Table II A or IIB the 3rd and 5 row, 6-15, the sequence of also expressing under the same conditions shown in 339-355 is capable is compared basic not reduction.

[0189.0.1.1] as the homologous compound of sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.1] to [0203.0.0.1]: see that [0190.0.0.0] is to [0203.0.0.0]

[0204.0.1.1] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 6-15,339-355 be capable, preferred Table II B the 7th row, 6-15 are capable, polypeptide or its segmental nucleic acid molecule of 339-355 shown in capable, described nucleic acid molecule is given each fine chemicals Threonine quantity in biological or its part increase;

(b) comprise, preferably comprise, the 339-355 capable as Table I the 5th or 7 row, capable, preferred Table I B the 7th row of 6-15,339-355,6-15 of mature form at least nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of each fine chemicals Threonine quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals Threonine quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals Threonine quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals Threonine quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals Threonine quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals Threonine quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as the primer shown in Table III A or IIIB the 7th row, 6-15,339-355 are capable or primer amplification from cDNA library or genomic library is obtained by using, and gives the increase of each fine chemicals Threonine quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals Threonine quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 6-15,339-355 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals Threonine quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals Threonine quantity increases, described peptide coding is capable as Table II the 5th or 7 row, capable, preferred Table II B the 7th row of 6-15,339-355,6-15, the structural domain of the polypeptide of 339-355 shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals Threonine quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7,6-15,339-355 is capable, preferred Table I B the 7th row, 6-15 is capable, nucleic acid molecule shown in 339-355 is capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, 6-15,339-355 is capable, preferred Table II B the 7th row, 6-15 is capable, at least the 15nt of the nucleic acid molecule of polypeptide shown in 339-355 is capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule do not form by Table I A or IB the 5th or 7 row, 6-15,339-355 by the sequence shown in capable.In one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 6-15, the 339-355 sequence shown in capable is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 6-15,339-355 capable shown in polypeptide of sequence.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in sequence at least 30%, 40%, 50% or 60% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as the peptide sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.Therefore, in one embodiment, nucleic acid molecule had at least one or more residues different shown in nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 6-15,339-355 were capable.Therefore, in one embodiment, the polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IB the 5th or 7 row, 6-15,339-355 are capable at least one or the different polypeptide of a plurality of amino acid.In another embodiment, Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in nucleic acid molecule coding schedule IIA or IIB the 5th or 7 row, 6-15, the 339-355 protein sequence shown in capable not.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.In another embodiment, protein sequence at least 30%, 40% shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable, 50% or 60% identical and with Table I A or IIB the 5th or 7 row, 6-15,339-355 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.1] to [0226.0.0.1]: see that [0205.0.0.0] is to [0226.0.0.0]

[0227.0.1.1] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except sequence or derivatives thereof shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of L-Methionin, L-methionine(Met) and/or L-Threonine biosynthetic pathway of at least one amino acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.1] to [0230.0.0.1]: see that [0228.0.0.0] is to [0230.0.0.0]

[0231.0.1.1] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened the degraded Threonine simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.1] to [0282.0.0.1]: see that [0232.0.0.0] is to [0282.0.0.0]

[0283.0.1.1] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that the fine chemicals Threonine increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-as proteinic antibody shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable, for example anti-antibody as polypeptide shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable, polypeptide (polypeptide for example of the present invention or its fragment) generation that it can comprise above-mentioned sequence or be made up of above-mentioned sequence by the standard technique utilization.Monoclonal antibody preferably.

[0284.0.0.1]: see [0284.0.0.0]

[0285.0.1.1] the present invention relates to have as sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable in one embodiment.

[0286.0.1.1] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, 6-15, consensus sequence shown in 339-355 is capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, 6-15, consensus sequence shown in 339-355 is capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can be by arbitrary aminoacid replacement.

[0287.0.0.1] to [0290.0.0.1]: see that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.1.1] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise and contain plant or microorganism specificity consensus sequence or by its polypeptide of forming.In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 6-15, sequence shown in 339-355 is capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 6-15, sequence shown in 339-355 is capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.

[0292.0.0.1]: see [0292.0.0.0]

[0293.0.1.1] the present invention relates to give the polypeptide that the fine chemicals Threonine increases in biological or its part in one embodiment, and described polypeptide is by nucleic acid molecule of the present invention or the employed nucleic acid molecule encoding of the present invention of the inventive method.

In one embodiment, polypeptide of the present invention have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help to form as the sequence of nucleic acid molecule encoding shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable.

[0294.0.1.1] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, 6-15, the polypeptide of activity of proteins shown in 339-355 is capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, 6-15, sequence shown in 339-355 is capable.

[0295.0.0.1] to [0297.0.0.1]: see that [0295.0.0.0] is to [0297.0.0.0]

The chemical of [0297.1.0.1] non-polypeptide of the present invention be for example do not have Table II A or IIB the 3rd, 5 or 7 row, 6-15,339-355 capable shown in the active polypeptide of polypeptide.

[0298.0.1.1] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II A or IIB the 5th or 7 row, 6-15,339-355, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.

[0299.0.1.1] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of nucleotide sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable.

[0300.0.1.1] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II A or IIB the 5th or 7,6-15,339-355 capable shown in sequence different.Therefore, this polypeptide contains with complete amino acid sequence homology as sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable and is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.1] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.1.1] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II A or IIB the 5th or 7 row, 6-15, the aminoacid sequence of the aminoacid sequence shown in 339-355 is capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.1]: see [0303.0.0.0]

[0304.0.1.1] operation nucleic acid molecule of the present invention may cause generation have basically as the active of polypeptide shown in Table II A or IIB, the 3rd row, 6-15,339-355 are capable and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.1] to [0308.0.0.1]: see that [0305.0.0.0] is to [0308.0.0.0]

[0309.0.1.1] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II A or IIB the 5th or 7 row, 6-15, be meant the polypeptide that has corresponding polypeptide of the present invention or be used for the amino acid sequence of polypeptide of the inventive method shown in 339-355 is capable, and be not shown in Table II A or IIB the 5th or 7 row, 6-15, the polypeptide that " non-polypeptide of the present invention " during 339-355 is capable or " other polypeptide " are meant the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with as Table II A or IIB the 5th or 7 be listed as, 6-15, polypeptide shown in 339-355 is capable is homology not basically, does not for example give described in the literary composition active or as Table II A or IIB, the 3rd row, 6-15, note shown in 339-355 is capable or known and from the protein of identical or different biology.In one embodiment, be not shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 " the non-polypeptide of the present invention " or " other polypeptide " in capable are not given fine chemicals in organism or its part increase.

[0310.0.0.1] to [0334.0.0.1]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.1.1] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of one of sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.1] to [0342.0.0.1]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.1.1] as describing, in order to cause effective reduction of expression, dsRNA and as Table I A or IB the 5th or 7 row, 6-15,339-355 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable or its homologue in a kind of organism.

[0344.0.0.1] to [0361.0.0.1]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.1.1] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or its part fine chemicals Threonine increases) carry out genetically modified any cell, the nucleic acid molecule of described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide (for example as Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable shown in polypeptide).Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or nucleic acid molecule of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.In one embodiment, the transgenosis that has as the polypeptide of polypeptide active shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable is meant in the text because genomic regulation and control or manipulation, activity as polypeptide shown in Table II A or IIB the 3rd row, 6-15,339-355 are capable (for example, having as polypeptide of sequence shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable) in cell or biological or its part improves.Example and the inventive method are described in above.

[0363.0.0.1]: see [0363.0.0.0]

[0364.0.1.1] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---combination of for example naturally occurring polypeptide promotor of the present invention and respective egg white matter encoding sequence---becomes transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.1] to [0382.0.0.1]: see that [0365.0.0.0] is to [0382.0.0.0]

[0383.0.1.1] is in order to prepare the hydroxyl fine chemicals, fine chemicals Threonine particularly, can use organic oxy-compound for example alcohol, hydroxyl organic acid, acetal contain carbonyl or the compound of carboxyl as currently known methods source of hydroxyl groups to be restored by this area.

[0384.0.0.1] to [0392.0.0.1]: see that [0384.0.0.0] is to [0392.0.0.0]

[0393.0.1.1] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that the fine chemicals Threonine increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify the nucleic acid molecule of particularly under lax stringent condition, hybridizing, and randomly separate full length cDNA clone or complete genome group clone as the nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable with nucleic acid molecule of the present invention;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce the fine chemicals Threonine, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.1] to [0398.0.0.1]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.1.1] in one embodiment, the present invention relates to identify the method for giving the compound that the fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under the fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving fine chemicals generation raising under these conditions.

Whether [0399.1.1.1] it is contemplated that by for example seeking the resistance of blocking-up fine chemicals Threonine synthetic medicine and observing this effect and depends on as the active of the polypeptide shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or its homologue or express, the fine chemicals Threonine that screens raising produces, for example with after the drug incubation with have low and high biology phenotype more much at one as activity of proteins shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable.

[0400.0.0.1] to [0430.0.0.1]: see that [0400.0.0.1] is to [0430.0.0.0]

[0431.0.1.1] embodiment 1: the SEQ IDNO:81 of ORF shown in the clones coding following table or in intestinal bacteria according to the other DNA polynucleotide of appended sequence list

[0432.0.1.1] uses the method for known good foundation (to consult as Sambrook, J. etc. (1989) " Molecular Cloning:A Laboratory Manual " Cold Spring HarborLaboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Mole-cular Biology ", John Wiley ﹠amp; Sons), with the SEQ IDNO:81 of ORF shown in the coding following table or according to the other DNA polynucleotide of appended sequence list be cloned into plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl Acad.Sci.USA, 75:3737-3741); PACYC177 (Change ﹠amp; Cohen (1978) J.Bacteriol.134:1141-1156); PBS series plasmid (pBSSK+, pBSSK-and other; Stratagene, LaJolla, USA) or clay such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J.Rosenthal, A., and Waterson, R.H. (1987) Gene 53:283-286) with at expression in escherichia coli.

[0433.0.0.1] to [0460.0.0.1]: see that [0433.0.0.0] is to [0460.0.0.0]

[0461.0.1.1] embodiment 10: clone SEQ ID NO:81 is used for expressing plant

[0462.0.0.1]: see [0462.0.0.0]

[0463.0.1.1] passes through pcr amplification SEQ ID NO:81 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.1] to [0466.0.0.1]: see that [0464.0.0.0] is to [0466.0.0.0]

[0467.0.1.1] selects following primer sequence for genes of SEQ ID NO:81:

I) forward primer SEQ ID NO:83:

ATGTCGTCCTTATCCACTTCATTTG

Ii) reverse primer SEQ ID NO:84:

TTAATTGTAACGGCTATATCTACTGG

[0468.0.0.1] to [0479.0.0.1]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.1.1] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:81

[0481.0.0.1] to [0513.0.0.1]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.1.1] selects as another kind of, as people such as Geigenberger (Plant Ceu﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect amino acid in the ethanol extraction.

As transformation arabidopsis thaliana as described in the embodiment 11.

The different mouse ear mustard of being analyzed belong to the following table that the results are shown in of (Arabidopsis) plant:

Table 1

??ORF	Note	Metabolite	??Min	??Max	Method
						??YFL050C	Two-trivalent inorganic cation transporter	Threonine	??1.193	??1.557	??GC
??YKR057W	Participate in the biological ribosomal protein that takes place and translate, be similar to the S21 ribosomal protein of rrna	Threonine	??1.34	??2.413	??GC
						??YIL150C	The S phase (DNA synthetic) is initial or finish necessary chromobindins	Threonine	??1.256	??4.186	??GC
??YNL046W	Possible endoplasmic reticulum albumen	Threonine	??1.178	??1.526	??GC
						??YNL120C	Do not characterize yet	Threonine	??1.44	??1.44	??LC
??b0186	Lysine decarboxylase	Threonine	??1.495	??3.277	??GC
						??b0730	Succinic thiokinase operon transcriptional and acyl are replied regulatory gene	Threonine	??1.531	??2.772	??LC
??b1829	Be defined as heat shock protein(HSP) with protease activity	Threonine	??1.174	??2.135	??GC
						??b2170	Sugar outflow transporter B	Threonine	??1.359	??1.792	??LC
??b0019	Na+/H+ antiport albumen	Threonine	??1.244	??1.44	??GC

Metabolite profile information:

??ORF	Metabolite	Method	??Min	??Max
					??b0464	Threonine	??GC	??1.23	??1.43
??b1360	Threonine	??GC	??1.16	??1.38
					??b1738	Threonine	??LC	??1.27	??4.61
??b1830	Threonine	??LC	??1.24	??1.43
					??b1896	Threonine	??LC+GC	??1.46	??2.08
??b2414	Threonine	??GC	??1.24	??1.46
					??b2552	Threonine	??GC	??1.17	??1.37
??b4004	Threonine	??GC	??1.17	??1.37
					??b2664	Threonine	??LC+GC	??1.29	??2.84
??b3074	Threonine	??LC	??1.31	??1.59
					??b2270	Threonine	??LC	??1.31	??1.59
??b3160	Threonine	??LC	??1.25	??1.56
					??b3231	Threonine	??GC	??1.17	??1.32
??b3462	Threonine	??GC	??1.18	??1.51
					??b3791	Threonine	??LC	??1.38	??1.44
??b3966	Threonine	??GC	??1.19	??1.47
					??YOR245C	Threonine	??GC	??1.18	??1.81

[0515.0.0.1]: to [0552.0.0.1]: see that [0515.0.0.0] is to [0552.0.0.0]

[0552.1.0.1]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF_ title	Metabolite	??Min	??Max
				??YIL150C	Threonine	??1.680	??2.977
??YKR057W	Threonine	??1.589	??5.593

Table 2 shows that Threonine has increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YIL150c or YKR057w.

In one embodiment, under the situation that yeast saccharomyces cerevisiae protein YIL150C or its homologue are enhanced as the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue in maize plant, preferably, giving the fine chemicals Threonine is increased between 1.68 and 2.977.

Under the situation that the activity of yeast saccharomyces cerevisiae protein YKR057W or the biological ribosomal protein that takes place and translate, be similar to the S21 ribosomal protein of participation rrna or its homologue is enhanced in maize plant, preferably, giving the fine chemicals Threonine is increased between 1.589 and 5.593.

[00552.2.0.1] sees [00552.2.0.0]

[0553.0.1.1]

1. produce the method for Threonine, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation Threonine.

2. produce the method for Threonine, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

(a) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable, and described nucleic acid molecule gives that Threonine quantity increases in biology or its part;

(b) contain just like IA or IB the 5th or 7 row, 6-15,339-355 the nucleic acid molecule of the nucleic acid molecule shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of Threonine quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of Threonine quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that Threonine quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 5th or 7 row, 6-15,339-355 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that fine chemicals Threonine quantity increases in biology or its part;

(g) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that Threonine quantity increases in biology or its part;

(h) nucleic acid molecule, its coding contain just like Table IV the 5th or 7 row, 6-15,339-355 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals Threonine quantity increase; With

(i) nucleic acid molecule, its can by under stringent hybridization condition with containing nucleic acid molecule (a) extremely

The suitable nucleic acid library of probe of one of sequence (k) or the screening of its fragment obtains, and give fine chemicals Threonine quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded is free or the bonded Threonine.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded Threonine of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

(a) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, 6-15,339-355 are capable, and described nucleic acid molecule gives that Threonine quantity increases in biology or its part;

(b) contain just like IA or IB the 5th or 7 row, 6-15,339-355 the nucleic acid molecule of the nucleic acid molecule shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of Threonine quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of Threonine quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that Threonine quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) nucleic acid molecule, it comprises by using Table III the 8th row, 6-15, the 339-355 primer nucleic acid molecule that amplifier nucleic acid molecule obtains from cDNA library or genomic library in capable, and gives that fine chemicals Threonine quantity increases in biology or its part;

(g) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that Threonine quantity increases in biology or its part;

(h) nucleic acid molecule, its coding contain just like Table IV the 8th row, 6-15,339-355 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals quantity increase; With

(i) nucleic acid molecule, its can by under stringent hybridization condition with containing nucleic acid molecule (a) extremely

The suitable nucleic acid library of probe of one of sequence (k) or the screening of its fragment obtains, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A or IB the 5th or 7 row, 6-15,339-355 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 9 or 10 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A or IIB the 5th or 7 be listed as, 6-15,339-355 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in Threonine quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part Threonine quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps Threonine level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the Threonine level that will measure or expression of polypeptides level and standard Threonine or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the Threonine generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of Threonine quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of Threonine quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the Threonine generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that Threonine increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce Threonine;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) the Threonine level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give Threonine level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the Threonine generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that Threonine quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce Threonine;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) the Threonine level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give Threonine level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that Threonine increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control Threonine level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

[0554.0.0.1] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.2]: in another embodiment, the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals.

[0001.0.0.2] to [0009.0.0.2]: see that [0.0.01.0.0.0] is to [0008.0.0.0]

[0009.0.2.2] as mentioned above, indispensable amino acid is that people and many Mammalss such as domestic animal are necessary.Tryptophane (L-tryptophane) is to have one of reactive amino acid most.At pH 4.0-6.0, the amino of tryptophane and aldehyde reaction produce Schiff alkali.On the other hand, if by acetylize amino is sealed, then tryptophane can obtain carboline derivative (carboline 1,2,3,4-tetrahydrochysene-carboline-3-carboxylic acid) with aldehyde reaction.Tryptophane plays a part unique in preventing infection, and this is that tryptophane is relative to be lacked because of comparing with other amino acid.In course of infection, health is induced and is produced the tryptophan metabolism enzyme, and it aggravates deficiency of tryptophan, to attempt to make the biology that is infecting [R.R.Brown, Y.Ozaki, S.P.Datta hungry to death, Deng, Implications of interferon-induced tryptophane catabolism incancer, auto-immune diseases and AIDS.In:Kynurenine and SerotoninPathways, R.Schwarcz etc., (editor), Plenum Press, New York, 1991].In most protein, comprise about 1% plant protein and 1.5% animal protein, tryptophane is least abundant indispensable amino acid.Though the minimum daily demand amout of tryptophane is 160mg for the women, is 250mg for the male sex, be recommended as 500-700mg to guarantee high-quality protein uptake.Actual tryptophane utilization is quite high.People use about 3.5g tryptophane, to make one day protein [J.C.Peters, Tryptophane Nutrition and Metabolism:anOverview.In:Kynurenine and Serotonin Pathways, R.Schwarcz etc., (editor), Plenum Press, New York, 1991].Realize balance by the liver recirculation of carrying out tryptophane from use (metabolism) protein.

Tryptophane in the diet is by the enteron aisle good absorption.In the round-robin tryptophane, about 10% is free in blood flow, and 90% is incorporated in on the white protein.Tryptophane binding site on the white protein also has affinity to free fatty acids (FFA), and therefore tryptophane is replaced when FFA raises, for example when fasting.

Though tryptophane is not the limiting amino acid of protein synthesis usually, is insufficient for the normal function of other tryptophane dependent pathway.Numerous research circuits have pointed to tryptophane at the central role of ingesting and other behavior is regulated.Tryptophane is rich in amino acid least usually in the liver total free aminoacids pond still not, and in the process of deprivation of food liver tryptophane-tRNA level than other indispensable amino acid faster [Q.R.Rogers that descends, The nutritional andmetabolic effects of amino acid imbalances.In:Protein Metabolism andNutrition, D.J.A.Cole (editor), Butterworths, London, 1976].Under the fasting situation and may be in wasting syndrome, it be the rate limiting acidic amino acid [Peters, 1991] of protein synthesis that tryptophane becomes.

[0010.0.0.2] and [0011.0.0.2]: see [0010.0.0.0] and [0011.0.0.0]

[0012.0.2.2] the objective of the invention is to develop the method for the economy of combination colour propylhomoserin, preferred L-tryptophane.The same with methionine(Met), Methionin and Threonine, tryptophane is one of the most normal amino acid that is restricted (depending on biology).

[0013.0.0.2]: see [0013.0.0.0]

[0014.0.2.2] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is tryptophane, preferred L-tryptophane.Therefore, in the present invention, term " fine chemicals " is meant " tryptophane " as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of tryptophane as used herein.

[0015.0.2.2] an embodiment, term " fine chemicals " meaning is meant tryptophane, preferred L-tryptophane.In whole specification sheets, term " fine chemicals " meaning is meant salt, ester or the acid amides of tryptophane, preferred L-tryptophane, its free form or is bonded to proteinic tryptophane, preferred L-tryptophane.In preferred embodiments, term " fine chemicals " meaning is meant tryptophane, preferred L-tryptophane or its salt of free form or is bonded to proteinic tryptophane, preferred L-tryptophane.

In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning is meant to have the active at least a chemical compound of above-mentioned fine chemicals.

[0016.0.2.2] therefore, the method that the present invention relates to may further comprise the steps:

(a) improve in non-human being or its one or more parts produce one or more following activity of proteins: YER173W, YGR104C, b0186, b0161, b0486, b1318, b2270, b3074, b3983 and/or YHR189W protein or have as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the protein of sequence of polypeptide of nucleic acid molecule encoding; With

(b) in allowing described biology, produce under the condition that fine chemicals is tryptophane or the fine chemicals that comprises tryptophane and make biological growth.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 the row, 16-18 is capable and/or 356-362 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) under the condition that allows generation fine chemicals, particularly tryptophane, make biological growth.

[0017.0.0.2] and [0018.0.0.2]: see [0017.0.0.0] and [0018.0.0.0]

The method that [0019.0.2.2] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare the fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in the capable and/or 356-362 of 16-18 is capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in the capable and/or 356-362 of 16-18 was capable.

[0020.0.2.2] be surprisingly found out that, at least a shown in Table II the 3rd row, 16-17 and/or 362 row yeast saccharomyces cerevisiae protein and/or at least aly give Threonine (or fine chemicals) the content raising of institute conversion plant as the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 3rd row, the 18th row and/or 356-361 are capable.

[0021.0.0.2]: see [0021.0.0.0]

The sequence of [0022.0.2.2] yeast saccharomyces cerevisiae YER173W has been published in Dietrich, Nature 387 (6632 supplementary issue), 78-81,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity be defined as " check position protein participates in the activation of dna damage and reduction division pachytene stage check position; Rad17p-Mec3p-Dc1p is loaded into the subunit of the clip loading bin (clamploader) on the DNA, the proteinic homologue of people and schizosaccharomyces pombe (S.pombe) Rad17; Rad24p ".Therefore, in one embodiment, method of the present invention comprises " Rad17p-Mec3p-Dc1p is loaded into the subunit of the clip loading bin on the DNA " or the purposes of Rad24p or Rad17 protein or its homologue from " the check position protein; participate in the activation of dna damage and reduction division pachytene stage check position " of yeast saccharomyces cerevisiae or its as shown in literary composition, it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The sequence of yeast saccharomyces cerevisiae YGR104C has been published in Thompson etc., Cell 73:1361-1375, and 1993, and its activity is defined as " yeast rna polymerase II repressor SRB5 ".Therefore, in one embodiment, the inventive method comprises the purposes with " rna plymerase ii repressor (yeast SRB5) " or its homologue, and it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The sequence of e. coli k12 b0186 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be lysine decarboxylase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary lysine decarboxylase or its homologue purposes, as described its to be used for producing fine chemicals in biological or its part be tryptophane, particularly increase tryptophane, the preferred quantity of the tryptophane of free or combining form.In one embodiment, the lysine decarboxylase activity is enhanced or produces in the methods of the invention, and for example the activity from colibacillary lysine decarboxylase or its homologue is enhanced or produces.

The sequence of e. coli k12 b0161 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises the active gene product of Helicobacterium as shown here (Helicobacter) serine protease superfamily, preferably has the active protein of pericentral siphon serine protease (heat shock protein) or its homologue, the purposes of protein or its homologue from e. coli k12 as shown here, its fine chemicals that is used for producing in biological or its part free or combining form is a tryptophane as described.

The sequence of e. coli k12 b0486 has been published in Blattner, and Science 277 (5331), 1453-1474, and 1997, and its activity is defined as amino acid/amine transporter (APC family).Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of membranin ybaT superfamily, preferably having a purposes of amino acid/amine transporter (APC family) active protein or its homologue, it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The sequence of e. coli k12 b1318 has been published in Blattner, and Science 277 (5331), 1453-1474, and 1997, and its activity is defined as HUCEP-8 (ATP-binding cassette superfamily).Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of inner membrane protein malK (having the ATP-binding cassette homology) superfamily, preferably having a purposes of the active protein of HUCEP-8 (ATP-binding cassette superfamily) or its homologue, it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The sequence of e. coli k12 b2270 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary protein b2270 or its homologue as shown here, and it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The sequence of e. coli k12 b3074 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having secretion molecular chaperones CsaA and/or methionyl-tRNA synthetic enzyme (forming dimeric) active gene product of superfamily, preferably having a purposes of protein or its homologue of tRNA synthase activity, it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The sequence of e. coli k12 b3983 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L12.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria ribosomal protein L11 superfamily, preferably having a purposes of the active protein of 50S ribosomal subunit protein matter L12 or its homologue, it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The sequence of yeast saccharomyces cerevisiae YHR189W has been published in and Goffeau, and Science 274 (5287), 546-547, and 1996 and Johnston, Nature 387 (6632 supplementary issue), 87-90,1997, and its activity is defined as the peptidyl tRNA lytic enzyme.Therefore, in one embodiment, the inventive method comprises having the active gene product of peptidyl tRNA lytic enzyme superfamily, preferably have a purposes of protein or its homologue of peptidyl tRNA hydrolytic enzyme activities from yeast saccharomyces cerevisiae as shown here, it is used for to be tryptophane, particularly to increase the quantity of free or combining form tryptophane at biological or its part generation fine chemicals as described.

The homologous compound (=homologue) of [0023.0.2.2] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.

Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with at least a Table I the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein, for example have by contain Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in capable and/or the 362nd row of Table II 16-17 in the polypeptide homologue of any one be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in the biology, and described homologue is from eukaryote.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 18th row and/or 356-361 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from bacterium.In one embodiment, Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from fungi.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 18th row and/or 356-361 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Proteobacteria.In one embodiment, Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Ascomycota.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 18th row and/or 356-361 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from yeast.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 18th row and/or 356-361 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the enterobacteria order.In one embodiment, Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 18th row and/or 356-361 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from enterobacteriaceae.In one embodiment, Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetes.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, the 18th row and/or 356-361 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Escherichia.In one embodiment, Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetaceae.In one embodiment, Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.

[0023.1.2.2] as Table II the 3rd row, 16-17 is capable and/or the 362nd row shown in the homologue of polypeptide can be by Table I the 7th row, 16-17 is capable and/or the 362nd row shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 16-17 is capable and/or the 362nd row shown in polypeptide.As the homologue of polypeptide shown in Table II the 3rd row, the 18th row and/or 356-361 are capable can be by Table I the 7th row, the 18th row and/or 356-361 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, the 18th row and/or 356-361 capable shown in polypeptide.

[0024.0.0.2]: see [0024.0.0.0]

[0025.0.2.2] is according to the present invention, tryptophan levels increases in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, any one proteinic biology or the enzyme activity shown in the capable and/or 356-362 of 16-18 is capable, if promptly with Table II the 3rd row, any one yeast belong protein shown in capable and/or the 362nd row of 16-17 compare and/or with Table II the 3rd row, any one e. coli k12 protein was compared shown in the 18th row and/or 356-361 were capable, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.2]: see [0025.1.0.0]

[0025.2.0.2]: see [0025.2.0.0]

[0026.0.0.2] to [0033.0.0.2]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.2.2] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or by Table I the 5th row, 16-18 is capable and/or 356-362 capable shown in the protein of nucleic acid molecule encoding or its homologue (as Table I the 7th row, 16-18 is capable and/or 356-362 capable shown in homologue) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows the fine chemicals quantity that increases.

[0035.0.0.2] to [0044.0.0.2]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.2.2] for example participates in dna damage and reduction division pachytene stage check position activatory check position protein at yeast saccharomyces cerevisiae protein YER173W or its homologue; Rad17p-Mec3p-Ddc1p is loaded into subunit or Rad24p or its homologue of the clip loading bin of DNA, for example under the situation that the activity of people or schizosaccharomyces pombe Rad17 (for example shown in Table I the 5th or 7 row, the 16th row) is enhanced, preferably, give fine chemicals be increased in 27% and 178% or higher between.

At yeast saccharomyces cerevisiae protein YGR104C or its homologue, for example under the activity situation about being enhanced of rna plymerase ii repressor (yeast SRB5) (shown in Table I the 5th or 7 row, the 17th row), preferably, give fine chemicals be increased in 32% and 84% or higher between.

Under the situation that the activity of e. coli k12 protein b0186 or lysine decarboxylase or its homologue (shown in Table I the 5th or 7 row, the 18th row) is enhanced, preferably, give fine chemicals and be increased between 32% and 146%.

Activity at e. coli k12 protein b0161 or its homologue, for example under the situation that Helicobacterium serine protease superfamily activity of proteins is enhanced, preferably, under the activity situation about being enhanced of the protein with pericentral siphon serine protease (heat shock protein) (shown in Table I the 5th or 7 row, the 356th row), give each fine chemicals, preferred color of choice propylhomoserin be increased in 93% and 278% or higher between.

Activity at e. coli k12 protein b0486 or its homologue, for example membranin ybaT superfamily activity of proteins, preferably have under the activity situation about being enhanced of the active protein of amino acid/amine transporter (APC family) (shown in Table I the 5th or 7 row, the 357th row), give each fine chemicals, preferred color of choice propylhomoserin be increased in 42% and 335% or higher between.

Activity at e. coli k12 protein b1318 or its homologue, for example inner membrane protein malK (having the ATP-binding cassette homology) superfamily activity, preferably have under the activity situation about being enhanced of the active protein of HUCEP-8 (ATP-binding cassette superfamily) (shown in Table I the 5th or 7 row, the 358th row), give each fine chemicals, preferred color of choice propylhomoserin be increased in 136% and 330% or higher between.

Activity at e. coli k12 protein b2270 or its homologue, for example under the activity situation about being enhanced of intestinal bacteria b2270 protein (shown in Table I the 5th or 7 row, the 359th row), give each fine chemicals, preferred color of choice propylhomoserin be increased in 33% and 79% or higher between.

Activity at e. coli k12 protein b3074 or its homologue, for example secrete molecular chaperones CsaA and/or methionyl-tRNA synthetic enzyme (forming dimeric) superfamily activity of proteins, preferably have under the activity situation about being enhanced of protein (shown in Table I the 5th or 7 row, the 360th row) of tRNA synthase activity, give each fine chemicals, preferred color of choice propylhomoserin be increased in 33% and 79% or higher between.

Activity at e. coli k12 protein b3983 or its homologue, for example the activity of intestinal bacteria ribosomal protein L11 superfamily, preferably have under the activity situation about being enhanced of the active protein of 50S ribosomal subunit protein matter L12 (shown in Table I the 5th or 7 row, the 361st row), give each fine chemicals, preferred color of choice propylhomoserin be increased in 33% and 387% or higher between.

Activity at yeast saccharomyces cerevisiae protein YHR189W or its homologue, peptidyl tRNA lytic enzyme superfamily activity for example, preferably have under the activity situation about being enhanced of protein (shown in Table I the 5th or 7 row, the 362nd row) of peptidyl tRNA hydrolytic enzyme activities, give each fine chemicals, preferred color of choice propylhomoserin be increased in 31% and 66% or higher between.

[0046.0.2.2] at yeast saccharomyces cerevisiae protein YER173W or its homologue, participates in dna damage and reduction division pachytene stage check position activatory check position protein in one embodiment; Rad17p-Mec3p-Ddc1p is loaded into subunit or Rad24p or its homologue of the clip loading bin of DNA, for example under the situation that the activity of people or schizosaccharomyces pombe Rad17 (for example shown in Table I the 5th or 7 row, the 16th row) is enhanced, preferably, giving fine chemicals and proline(Pro) increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YGR104C or its homologue, for example under the activity situation about being enhanced of rna plymerase ii repressor (yeast SRB5) (shown in Table I the 5th or 7 row, the 17th row), preferably, giving fine chemicals and L-glutamic acid increases.

In one embodiment, at e. coli k12 protein b0186 or its homologue, for example under the activity situation about being enhanced of lysine decarboxylase or its homologue (shown in Table I the 5th or 7 row, the 18th row), preferably, giving fine chemicals and methionine(Met) increases.

In one embodiment, activity at e. coli k12 protein b0161 or its homologue is enhanced, for example Helicobacterium serine protease superfamily activity of proteins is enhanced, preferably, have under the activity situation about being enhanced of the active protein of pericentral siphon serine protease (heat shock protein) (shown in Table I the 5th or 7 row, the 356th row), giving each fine chemicals, preferred color of choice propylhomoserin and other amino acid increases.

In one embodiment, activity at e. coli k12 protein b0486 or its homologue is enhanced, for example membranin ybaT superfamily protein, preferably have under the activity situation about being enhanced of the active protein of amino acid/amine transporter (APC family) (shown in Table I the 5th or 7 row, the 357th row), giving each fine chemicals, preferred color of choice propylhomoserin and other amino acid increases.

In one embodiment, activity at e. coli k12 protein b1318 or its homologue is enhanced, for example inner membrane protein malK (having the ATP-binding cassette homology) superfamily, preferably have under the activity situation about being enhanced of the active protein of HUCEP-8 (ATP-binding cassette superfamily) (shown in Table I the 5th or 7 row, the 358th row), giving each fine chemicals, preferred color of choice propylhomoserin and other amino acid increases.

In one embodiment, activity at e. coli k12 protein b2270 or its homologue is enhanced, for example under the activity situation about being enhanced of transcriptional (shown in Table I the 5th or 7 row, the 359th row), giving each fine chemicals, preferred color of choice propylhomoserin and other amino acid increases.

In one embodiment, activity at e. coli k12 protein b3074 or its homologue is enhanced, for example tRNA synthetic enzyme or its homologue, under the activity situation about being enhanced of transcriptional (shown in Table I the 5th or 7 row, the 360th row), giving each fine chemicals, preferred color of choice propylhomoserin and other amino acid increases.

In one embodiment, activity at e. coli k12 protein b3983 or its homologue is enhanced, for example intestinal bacteria ribosomal protein L11 superfamily, preferably have under the activity situation about being enhanced of the active protein of 50S ribosomal subunit protein matter L12 (shown in Table I the 5th or 7 row, the 361st row), giving each fine chemicals, preferred color of choice propylhomoserin and other amino acid increases.

In one embodiment, activity at yeast saccharomyces cerevisiae protein YHR189W or its homologue is enhanced, for example peptidyl tRNA lytic enzyme superfamily, preferably have under the activity situation about being enhanced of protein (shown in Table I the 5th or 7 row, the 362nd row) of peptidyl tRNA hydrolytic enzyme activities, giving each fine chemicals, preferred color of choice propylhomoserin and other amino acid increases.

[0047.0.0.2] and [0048.0.0.2]: see [0047.0.0.0] and [0048.0.0.0]

[0049.0.2.2] has to give and improves fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, the polypeptide of consensus sequence shown in the capable and/or 356-362 of 16-18 is capable, perhaps as Table II the 5th or 7 row, its function homologue described in polypeptide or the literary composition shown in the capable and/or 356-362 of 16-18 is capable, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the capable and/or 356-362 of 16-18 is capable) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.2.2] for the purposes of the present invention, term " tryptophane " and " L-tryptophane " also comprise corresponding salt, for example tryptophane hydrochloride or tryptophane vitriol.Preferably, the term tryptophane is intended to comprise term L-tryptophane.

[0051.0.0.2] and [0052.0.0.2]: see [0051.0.0.0] and [0052.0.0.0]

[0053.0.2.2] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein give the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide) express and increase, have the activity of the raising fine chemicals described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of the raising tryptophane described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give the raising tryptophane that has described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give the raising tryptophane that has described in the literary composition active, by the used polypeptide of the coded protein of the used nucleic acid molecule of nucleic acid molecule of the present invention or the present invention or polypeptide of the present invention or the present invention (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide) express and improve;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give the raising tryptophane that has described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide) express and improve;

(f) express the transgenosis of coded protein, described protein give the raising tryptophane that has described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have the raising tryptophane described in the literary composition active, by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide);

(h) by add positive Expression element or remove negative Expression element improve code book invention polypeptide or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.2.2] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improve coded protein or have as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide) give tryptophane after active expression of polypeptides or the activity and increase.

[0055.0.0.2] to [0071.0.0.2]: see that [0055.0.0.0] is to [0071.0.0.0]

[0072.0.2.2] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has chorismic acid, anthralinicacid, N-5 '-Phosphoribosyl-anthranilic acid, 1-(o-carboxyl phenylamino)-1-deoxyribulose 5-phosphoric acid, 1-(indol-3-yl)-glycerol-3-phosphate and 5-dihydroxyphenylethylamine except tryptophane.

[0073.0.2.2] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or the used polypeptide of the inventive method or its homologue (for example Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or bonded fine chemicals by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation, and optional other dissociates and/or in conjunction with amino acid.

[0074.0.0.2] to [0084.0.0.2]: see that [0074.0.0.0] is to [0084.0.0.0]

[0085.0.2.2] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.2] and [0087.0.0.2]: see [0086.0.0.0] and [0087.0.0.0]

[0088.0.2.2] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified aminoacids content.Often be subject to minority indispensable amino acid (as Methionin, Threonine or methionine(Met) or tryptophane) owing to for example be used to raise the nutritive value of the plant of monogastric animal, so this is very important for the plant breeder.

[0088.1.0.2] to [0097.0.0.2]: see that [0088.1.0.0] is to [0097.0.0.0]

[0098.0.2.2] in preferred embodiments, the fine chemicals Threonine be produce according to the present invention and carry out isolating where necessary.It is favourable as methionine(Met), Methionin and/or aminoacid mixture that the method according to this invention produces other amino acid.

[0099.0.0.2] to [0102.0.0.2]: see that [0099.0.0.0] is to [0102.0.0.0]

[0103.0.2.2] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) the preferred nucleic acid molecule of encoding mature form polypeptide at least, described polypeptide have as Table II the 5th or 7 row, 16-18 is capable and/or the sequence of 356-362 shown in capable;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I the 5th or 7 row, 16-18 is capable and/or the sequence of 356-362 shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use have as Table III the 7th row, 16-18 is capable and/or 356-362 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, 16-18 is capable and/or 356-362 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.0.0.] in one embodiment, be used for the nucleic acid molecule of the inventive method and Table I A the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the nucleic acid molecule that is used for the inventive method not by Table I A the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence form.In one embodiment, be used for the nucleic acid molecule of the inventive method and Table I A the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II A the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide of sequence.

[00103.2.0.0.] in one embodiment, be used for the nucleic acid molecule of the inventive method and Table I B the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the nucleic acid molecule that is used for the inventive method not by Table I B the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence form.In one embodiment, be used for the nucleic acid molecule of the inventive method and Table I B the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide of sequence.

[0104.0.2.2] in one embodiment, nucleic acid molecule of the present invention or be used for the nucleic acid molecule of the inventive method and Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention or the nucleic acid that is used for the inventive method not by Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide of sequence.

[0105.0.0.2] to [0107.0.0.2]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.2.2] method of the present invention and had as Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in the capable and/or 356-362 of 16-18 is capable, from being listed as Table II the 5th or 7, aminoacid sequence shown in the capable and/or 356-362 of 16-18 is capable is derived or from containing just like Table IV the 7th row, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in the capable and/or 356-362 of 16-18 is capable, perhaps its coding has as Table II the 3rd, 5 or 7 row, active or the bioactive polypeptide of polypeptidase shown in the capable and/or 356-362 of 16-18 is capable or for example give derivative or the homologue that fine chemicals increases behind its expression or active the increasing.

[0109.0.0.2]: see [0109.0.0.0]

[0110.0.2.2] help the inventive method and coding have polypeptide of the present invention be used for the polypeptide of the inventive method or be used for process of the present invention polypeptide (for example Table II the 5th row, 16-18 is capable and/or 356-362 capable shown in protein or by Table I the 5th row, 16-18 is capable and/or 356-362 capable shown in coded protein or its homologue of nucleic acid molecule, as Table II the 7th row, 16-18 is capable and/or 356-362 capable shown in) nucleic acid molecule of active polypeptide can determine from generally open database.

[0111.0.0.2]: see [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.2.2] the inventive method is the isolated nucleic acid sequences form, its coding have as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in polypeptide active polypeptide or have as Table II the 5th and 7 row, 16-18 is capable and/or 356-362 capable shown in the polypeptide of peptide sequence, and give tryptophane and increase.

[0113.0.0.2] to [120.0.0.2]: see that [0113.0.0.0] is to [0120.0.0.0]

[0121.0.2.2] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in peptide sequence or its functional homologue as herein described the difference of one or more amino acid moleculars is arranged, described artificial sequence is preferably given aforementioned activity, and promptly giving fine chemicals after improving its activity increases.

[0122.0.0.2] to [0127.0.0.2]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.2.2] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 16-18 is capable and/or the primer of 356-362 shown in capable to) can based on as Table I the 5th or 7 row, 16-18 is capable and/or the sequence of 356-362 shown in capable or from as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence deutero-sequence produce.

[0129.0.2.2] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of the inventive method (sequence of polypeptide particularly of the present invention or the used polypeptide of the inventive method).Indicate in the shown in the drawings comparison of the conservative region of polypeptide of the present invention or the used polypeptide of the inventive method.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, 16-18 is capable and/or 356-362 capable shown in consensus sequence from described comparison.

[0130.0.2.2] to [0138.0.0.2]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.2.2] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving tryptophane increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence hybridization, and coding is expressed and to be had the active peptide of the tryptophane of increasing.

[0140.0.0.2] to [0146.0.0.2]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.2.2] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.2.2] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 5th or 7 row, nucleotide sequence or its funtion part homology were at least about 30% shown in the capable and/or 356-362 of 16-18 was capable, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases fine chemicals.

[0149.0.2.2] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in one of nucleotide sequence or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (increasing as giving fine chemicals) and as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 5th or 7 row, 16-18 is capable and/or the protein of 356-362 shown in capable.

[0149.1.2.2] randomly, with Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd row, 16-18 is capable and/or the protein of 356-362 shown in capable is known activity or is used for these protein of note.

[0150.0.2.2] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor can only contain Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving tryptophane when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in one of sequence sense strand, Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of nucleotide sequence of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 16-18 is capable and/or 356-362 capable shown in the right PCR of primer will produce as Table I the 5th or 7 be listed as, 16-18 is capable and/or 356-362 capable shown in the fragment of polynucleotide sequence.

[0151.0.0.2]: see [0151.0.0.0]

[0152.0.2.2] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of tryptophane, specifically be included in plant or the microorganism as described above or the activity of raising tryptophane as be shown in the examples.

[0153.0.2.2] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing tryptophane.In one embodiment, as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in protein or its part have Table II the 3rd row for example, 16-18 is capable and/or 356-362 capable shown in the activity of polypeptide.

[0154.0.2.2] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving fine chemicals increases.

[0155.0.0.2] and [0156.0.0.2]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.2.2] the present invention relate in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology tryptophane increase), for example comprise as Table IV the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in consensus sequence polypeptide or as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment as Table IV the 5th or 7 row, 16-18 is capable and/or the consensus sequence of 356-362 shown in capable or as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as Table IV the 7th row, 16-18 is capable and/or the consensus sequence of 356-362 shown in capable or as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 16-18 is capable and/or the sequence of 356-362 shown in capable.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in nucleic acid molecule identical.

[0158.0.0.2] to [0160.0.0.2]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.2.2] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.2]: see [0162.0.0.0]

[0163.0.2.2] preferably, under stringent condition with Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.2]: see [0164.0.0.0]

[0165.0.2.2] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.2] and [0167.0.0.2]: see [0166.0.0.0] and [0167.0.0.0]

[0168.0.2.2] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or 356-362 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or the aminoacid sequence of 356-362 shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, sequence shown in the capable and/or 356-362 of 16-18 is capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, one of sequence shown in the capable and/or 356-362 of 16-18 is capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, sequence shown in the capable and/or 356-362 of 16-18 is capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, sequence shown in the capable and/or 356-362 of 16-18 is capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.2] to [0172.0.0.2]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.2.2] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:732 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:732 sequence before use.

[0174.0.0.2]: see [0174.0.0.0]

[0175.0.2.2] for example, the sequence that has 80% homology at protein level and SEQ ID NO:733 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQID NO:733 sequence.

[0176.0.2.2] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in the capable and/or 356-362 of 16-18 was capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in the capable and/or 356-362 of 16-18 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in the capable and/or 356-362 of 16-18 is capable has essentially identical character and discerns.

[0177.0.2.2] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, the functional equivalent that nucleotide sequence obtained shown in the capable and/or 356-362 of 16-18 was capable be listed as according to of the present invention as Table II the 5th or 7,16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, one of polypeptide had at least 30% shown in the capable and/or 356-362 of 16-18 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, the polypeptide of the essentially identical character of polypeptide shown in the capable and/or 356-362 of 16-18 is capable.

[0178.0.0.2]: see [0178.0.0.0]

[0179.0.2.2] can by to the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in) in introduce replacement, interpolation or the disappearance of one or more Nucleotide, and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th is listed as, 16-18 is capable and/or 356-362 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 introduces sudden change in the encoding sequence of sequence shown in capable.

[0180.0.0.2] to [0183.0.0.2]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.2.2] is employed to have as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in the capable and/or 356-362 of 16-18 is capable, perhaps come Table II the 5th or 7 row freely, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, the homologous compound of the nucleotide sequence of sequence shown in the capable and/or 356-362 of 16-18 is capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.2.2] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, 16-18 is capable and/or the sequence of 356-362 shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, 16-18 is capable and/or the arbitrary sequence of 356-362 shown in capable in other nucleotide sequence of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, 16-18 is capable and/or the sequence of 356-362 shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.2.2] also preferred the inventive method comprise as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or 356-362 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the coded polynucleotide that are used for the inventive method with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or 356-362 capable shown in sequence identical.

[0187.0.2.2] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or 356-362 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or 356-362 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.2.2] still has the polypeptide basic enzyme activity of the present invention (being its active basic reduction that do not have) of giving fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, polypeptide active shown in the capable and/or 356-362 of 16-18 is capable, preferably with Table II the 3rd and 5 row, shown in the capable and/or 356-362 of 16-18 is capable and the sequence of expressing under the same conditions compare and do not reduce substantially.

In one embodiment, polypeptide of the present invention is a homologue, its comprise as Table II B the 7th row, 16-18 is capable and/or 356-362 capable shown in sequence or especially form.

[0189.0.2.2] as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the homologous compound of sequence, or deutero-as Table II the 5th or 7 row, 16-18 is capable and/or the homologue of the sequence of 356-362 shown in capable also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.2] to [0203.0.0.2]: see that [0190.0.0.0] is to [0203.0.0.0]

[0204.0.2.2] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or polypeptide or its segmental nucleic acid molecule of 356-362 shown in capable, described nucleic acid molecule is given fine chemicals quantity in biological or its part increase;

(b) comprise, preferably comprise mature form at least as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or nucleic acid molecule or its segmental nucleic acid molecule of 356-362 shown in capable, described nucleic acid molecule is given the increase of fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 7th row, 16-18 is capable and/or the primer of 356-362 shown in capable or primer obtain amplification from cDNA library or genomic library nucleic acid molecule, and give the increase of fine chemicals quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 16-18 is capable and/or 356-362 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described peptide coding as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, 16-18 is capable and/or the structural domain of the polypeptide of 356-362 shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7,16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, nucleic acid molecule shown in the capable and/or 356-362 of 16-18 is capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table II B the 7th row, at least the 15nt of the nucleic acid molecule of polypeptide shown in the capable and/or 356-362 of 16-18 is capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby, the nucleic acid molecule of preferred (a) to (l) by one or more Nucleotide be different from as Table I A the 5th or 7 row, 16-18 is capable and/or the sequence of 356-362 shown in capable.In one embodiment, nucleic acid molecule not by Table I A or IB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in and the sequence described form.In one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 16-18 is capable and/or the sequence of 356-362 shown in capable is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide of sequence.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence at least 30%, 40%, 50% or 60% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as Table II A or IIB the 5th or 7 row, 16-18 is capable and/or the peptide sequence of 356-362 shown in capable.Therefore, in one embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in nucleic acid molecule have at least one or more residues different.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IB the 5th or 7 row, 16-18 is capable and/or the polypeptide of 356-362 shown in capable at least one or the different polypeptide of a plurality of amino acid.In another embodiment, Table I A or IB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 16-18 is capable and/or the protein sequence of 356-362 shown in capable.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II A or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in protein sequence at least 30%, 40%, 50% or 60% identical and with Table I A or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.2] to [0226.0.0.2]: see that [0205.0.0.0] is to [0226.0.0.0]

[0227.0.2.2] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the sequence or derivatives thereof, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of L-Methionin, L-Threonine and/or L-methionine(Met) or L-tryptophane biosynthetic pathway of at least one amino acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.2] to [0230.0.0.2]: see that [0228.0.0.0] is to [0230.0.0.0]

[0231.0.0.2] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened the degraded tryptophane simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.2] to [0282.0.0.2]: see that [0232.0.0.0] is to [0282.0.0.0]

[0283.0.2.2] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in proteinic antibody, for example anti-as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the antibody of polypeptide, polypeptide (polypeptide for example of the present invention or its fragment) generation that it can comprise above-mentioned sequence or be made up of above-mentioned sequence by the standard technique utilization.Preferably specificity in conjunction with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the monoclonal antibody of polypeptide.

[0284.0.0.2] sees [0284.0.0.0]

[0285.0.2.2] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence or by as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.2.2] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in the capable and/or 356-362 of 16-18 is capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in the capable and/or 356-362 of 16-18 is capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, 16-18 is capable and/or 356-362 capable shown in the polypeptide of consensus sequence.

[0287.0.0.2] to [290.0.0.2]: see that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.2.2] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise and contain plant or microorganism specificity consensus sequence or by its polypeptide of forming.In one embodiment, described polypeptide of the present invention by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 16-18 is capable and/or the sequence of 356-362 shown in capable.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in the capable and/or 356-362 of 16-18 is capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in the capable and/or 356-362 of 16-18 is capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence form.

[0292.0.0.2]: see [0292.0.0.0]

[0293.0.2.2] the present invention relates to give the polypeptide that each fine chemicals increases in biological or its part in one embodiment, and described polypeptide is by nucleic acid molecule of the present invention or the employed nucleic acid molecule encoding of the inventive method.

In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in other sequence of sequence phase region.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence form.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help as Table I A or IB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the sequence of nucleic acid molecule encoding form.

[0294.0.2.2] in one embodiment, the present invention relates to have as Table II the 3rd row, the polypeptide of activity of proteins shown in the capable and/or 356-362 of 16-18 is capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in the capable and/or 356-362 of 16-18 is capable.

[0295.0.0.2] to [0297.0.0.2]: see that [0295.0.0.0] is to [0297.0.0.0]

The chemical of [0297.1.0.2] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 16-18 is capable and/or 356-362 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.

[0298.0.2.2] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the identical aminoacid sequence of sequence.

[0299.0.2.2] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in one of nucleotide sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.2.2] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,16-18 is capable and/or 356-362 capable shown in sequence different.Therefore, this polypeptide contain with as Table II A or IIB the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.2]: see [0301.0.0.0]

The biologically-active moiety of [0302.0.2.2] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence shown in the capable and/or 356-362 of 16-18 is capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.2]: see [0303.0.0.0]

[0304.0.2.2] operation nucleic acid molecule of the present invention may cause generation have basically as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.2] to [0306.0.0.2]: see that [0305.0.0.0] is to [0306.0.0.0]

[00306.1.2.2] preferably, compound is the composition of the tryptophane that comprises tryptophane or recovery (particularly free or with the fine chemicals of protein bound form).

[0307.0.0.2]: to [0308.0.0.2]: see that [0305.0.0.0] is to [03086.0.0.0]

[0309.0.2.2] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, be meant the polypeptide that has corresponding polypeptide of the present invention or be used for the amino acid sequence of polypeptide of the inventive method shown in the capable and/or 356-362 of 16-18 is capable, be not listed as and be shown in Table II the 5th or 7, the polypeptide that " non-polypeptide of the present invention " or " other polypeptide " during the capable and/or 356-362 of 16-18 is capable is meant the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with as Table II the 5th or 7 be listed as, polypeptide shown in the capable and/or 356-362 of 16-18 is capable is homology not basically, for example do not give described in the literary composition active or as Table II the 3rd be listed as, note shown in the capable and/or 356-362 of 16-18 is capable or known and from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, 16-18 is capable and/or " the non-of the present invention polypeptide " or " other polypeptide " of 356-362 in capable do not given fine chemicals in organism or its part increase.

[0310.0.0.2] to [334.0.0.2]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.2.2] confirmed the dsRNAi method to reduce as Table II the 5th or 7 row, 16-18 is capable and/or the expression of the nucleotide sequence of 356-362 shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of one of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.2] to [0342.0.0.2]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.2.2] as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.2] to [0361.0.0.2]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.2.2] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or its part fine chemicals increases) carry out genetically modified any cell, used nucleic acid molecule, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or the code book invention polypeptide of described nucleic acid nucleic acid molecule for example of the present invention, the inventive method (for example as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide) nucleic acid molecule.Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of used polypeptide of polypeptide of the present invention or the inventive method or nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor improves, for example because the expression of research object of the present invention or specific activity raising in cell or biological or its part.In one embodiment, have as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part as Table II the 3rd row, 16-18 is capable and/or 356-362 capable shown in polypeptide (for example, have as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide of sequence) activity improve.Example and the inventive method are described in above.

[0363.0.0.2] to [0382.0.0.2]: see that [0365.0.0.0] is to [0382.0.0.0]

[0383.0.2.2] is in order to prepare the fine chemicals of aromatic-containing compound, fine chemicals particularly, can use organic aromatic compound for example benzene, naphthalene, indoles, pyrroles, furans (furen), oxazole, imidazoles, thiophene, pyrans, pyrimidine or other organic aromatic compound for example phenylformic acid and chorismic acid, shikimic acid, aminobenzoic acid, kynurenic acid or pyridoxal (pyridoxidal) are originated as aromatic ring.

[0384.0.0.2]: see [0384.0.0.0]

[0385.0.2.2] particularly contains for example fermented liquid of L-tryptophane, L-methionine(Met), L-Threonine and/or L-Methionin with what this kind approach obtained, and dry matter content accounts for 7.5 to 25% of weight usually.At least when finishing, particularly passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means that the concentration that can utilize sugar in the fermention medium during this period of time remains on or be reduced to 0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant) or these methods or partly from fermented liquid, shift out or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.0.2] to [392.0.0.2]: see that [0386.0.0.0] is to [0392.0.0.0]

[0393.0.2.2] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 16-18 is capable and/or 356-362 is capable, preferred Table I B the 7th row, 16-18 is capable and/or the nucleic acid molecule of 356-362 shown in capable hybridized under lax stringent condition nucleic acid molecule, and randomly separates full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.2] to [0399.0.0.2]: see that [0394.0.0.0] is to [0399.0.0.0]

[0399.1.2.2] it is contemplated that by for example seeking the resistance of blocking-up fine chemicals synthetic medicine and observing whether this effect depends on as Table II the 5th or 7 row, 16-18 is capable and/or the polypeptide of 356-362 shown in capable or its homologue active or express, the fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.2] to [0416.0.0.2]: see that [0400.0.0.0] is to [0416.0.0.0]

[0417.0.2.2] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of amino acid biosynthetic pathway is had resistance.Particularly, the expression excessively of polypeptide of the present invention can make amino acid, particularly fine chemicals synthetic weedicide in the described plant of the anti-blocking-up of plant.The example of amino acid synthetic weedicide is for example sulfonylurea and imidazolidinone weedicide in the blocking-up plant, the first step in these weedicide catalysis branched-chain amino acid biosynthesizing.

[0418.0.0.2] to [0423.0.0.2]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.2.2] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, agonist with the inventive method evaluation, the nucleic acid molecule of identifying with the inventive method can be used to produce fine chemicals or produce fine chemicals and one or more other amino acid, particularly methionine(Met), Threonine, L-Ala, glutamine, L-glutamic acid, Xie Ansuan, l-asparagine, phenylalanine, leucine, proline(Pro), tryptophane, tyrosine, Isoleucine and arginine.Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing the fine chemicals of biological or its part (as cell).

[0425.0.0.2] to [0430.0.0.2]: see that [0425.0.0.0] is to [0430.0.0.0]

[0431.0.2.2] embodiment 1: clone SEQ ID No:732 in intestinal bacteria

[0432.0.0.0] to [0460.0.0.2]: see that [0433.0.0.0] is to [0460.0.0.0]

[0461.0.2.2] embodiment 10: clone SEQ ID NO:732 is used for expressing plant

[0462.0.0.2] sees [0462.0.0.0]

[0463.0.2.2] passes through pcr amplification SEQ ID NO:732 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.2] to [0466.0.0.2]: see that [0464.0.0.0] is to [0466.0.0.0]

[0467.0.2.2] selects following primer sequence for genes of SEQ ID NO:732:

I) forward primer (SEQ ID No:734):

ATGGATAGTACGAATTTGAACAAACG

Ii) reverse primer (SEQ ID N0:735):

TTAGAGTATTTCCAGATCTGAATCTG

[0468.0.0.2] to [0479.0.0.2]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.2.2] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:732

[0481.0.0.2] to [0513.0.0.2]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.2.2] selects as another kind of, as people such as Geigenberger (Plant Cell﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect amino acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Note	Metabolite	??Min	??Max	Method
						??YER173W	Participate in dna damage and reduction division pachytene stage check position activatory check position protein	Tryptophane	??1.27	??2.78	??LC
??YGR104C	Yeast rna polymerase II repressor SRB5; RNA polymerase B repressor SRB5	Tryptophane	??1.32	??1.84	??LC
						??b0186	Lysine decarboxylase	Tryptophane	??1.32	??2.46	??LC

Table 1b

??ORF	??MetChemID	Metabolite	Method	??Min	??Max
						??b0161	??10000035	Tryptophane	??LC	??1.93	??3.78

??ORF	??MetChem?ID	Metabolite	Method	??Min	??Max
						??b0486	??10000035	Tryptophane	??LC	??1.42	??4.35
??b1318	??10000035	Tryptophane	??LC	??2.36	??4.30
						??b2270	??10000035	Tryptophane	??LC	??1.33	??1.79
??b3074	??10000035	Tryptophane	??LC	??1.33	??1.79
						??b3983	??10000035/30000016	Tryptophane	??LC+GC	??1.33	??4.87
??YHR189W	??10000035	Tryptophane	??LC	??1.31	??1.66

[0515.0.0.2] to [0552.2.0.2]: see that [0515.0.0.0] is to [0552.2.0.0]

[0553.0.2.2]

1. produce the method for tryptophane, it comprises:

(a) improve in non-human being or its one or more parts or produce as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation tryptophane.

2. produce the method for tryptophane, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

(a) nucleic acid molecule, coding as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part tryptophane quantity to be increased;

(b) contain just like Table I the 5th or 7 row, 16-18 is capable and/or the nucleic acid molecule of the nucleic acid molecule of 356-362 shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of tryptophane quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of tryptophane quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that tryptophane quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) nucleic acid molecule, it comprise by use as Table III the 7th row, 16-18 is capable and/or 356-362 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that fine chemicals quantity increases in biology or its part;

(g) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that tryptophane quantity increases in biology or its part;

(b) nucleic acid molecule, its coding contain just like Table IV the 7th row, 16-18 is capable and/or 356-362 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals quantity increase; With

(i) nucleic acid molecule, its can by under stringent hybridization condition with containing nucleic acid molecule (a) extremely

The suitable nucleic acid library of probe of one of sequence (k) or the screening of its fragment obtains, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded tryptophane.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded tryptophane of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

(a) nucleic acid molecule, coding as Table II the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part tryptophane quantity to be increased;

(b) contain just like Table I the 5th or 7 row, 16-18 is capable and/or the nucleic acid molecule of the nucleic acid molecule of 356-362 shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of tryptophane quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of tryptophane quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that tryptophane quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) nucleic acid molecule, it comprise by use as Table III the 7th row, 16-18 is capable and/or 356-362 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that tryptophane quantity increases in biology or its part;

(g) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that tryptophane quantity increases in biology or its part;

(h) nucleic acid molecule, its coding contain just like Table IV the 7th row, 16-18 is capable and/or 356-362 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals quantity increase; With

(i) nucleic acid molecule, its can by under stringent hybridization condition with containing nucleic acid molecule (a) extremely

The suitable nucleic acid library of probe of one of sequence (k) or the screening of its fragment obtains, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, 16-18 is capable and/or 356-362 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 9 or 10 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 16-18 is capable and/or 356-362 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in tryptophane quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part tryptophane quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps tryptophan levels or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the tryptophan levels that will measure or expression of polypeptides level and reference colour propylhomoserin or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the tryptophane generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of tryptophane quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of tryptophane quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the tryptophane generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that tryptophane increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce tryptophane;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) tryptophan levels in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give tryptophan levels raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the tryptophane generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that tryptophane quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce tryptophane;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) tryptophan levels in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give tryptophan levels raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that tryptophane increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control tryptophan levels at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 17, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make the anti-tryptophane synthetic weedicide that suppresses of plant.

[0554.0.0.2] summary: see [0554.0.0.0]:

Produce the method for fine chemicals

[0000.0.0.3] the present invention relates to produce the another kind of method of following defined fine chemicals and corresponding embodiment as described below in another embodiment.

[0001.0.0.3] to [0008.0.0.3]: see that [0001.0.0.0] is to [0008.0.0.0]

[0009.0.3.3] as mentioned above, indispensable amino acid is that people and many Mammalss such as domestic animal are necessary.Branched-chain amino acid (BCAA) leucine, Isoleucine and Xie Ansuan belong to human diet the amino acid in the middle of requisite 9 seed amino acids.BCAA accounts for the requisite amino acid whose 35-40% of body protein kind diet, and 14% of total amino acid (Ferrando etc., (1995) Oral branched chain amino acids decrease whole-body proteolysis.J.Parenter.Enteral Nutr.19:47-54.13) in the skeletal muscle.They are shared common film movement system and are used to change the enzyme (Block of ammonia and irreversible oxidation, K.P. (1989) Interactions among leucine, isoleucine, and valine with special reference-the branched chain amino acidantagonism.In:Absorption and utilizes of Amino Acids (Friedman, M. compiles), 229-244 page or leaf, CRC Press, Boca Raton, FL. and Champe, P.C.﹠amp; Harvey, R.A. (1987) Amino acids:metabolism of carbon atoms.In:Biochemistry (Champ, P.C.﹠amp; Harvery, P.A. compiles), the 242-252 page or leaf, J.B.Lippincott, Philadelphia, PA.).In addition, for the patient who suffers from maple sugar uraturia disease (MSUD), be necessary to reduce the picked-up of these branched-chain amino acids.

The diet source of branched-chain amino acid is mainly from animal proteinum and vegetable-protein.Branched-chain amino acid (BCAA) leucine, Isoleucine and Xie Ansuan are restrictive for many mammiferous growths.Therefore, in the food feed of meat chicken, laying hen, turkey, pig or ox, replenish branched-chain amino acid.

[0010.0.0.3] sees [0010.0.0.0]

[0011.0.0.3] sees [0011.0.0.0]

[0012.0.3.3] the objective of the invention is to develop the method for the economy of synthesizing leucine and/or Isoleucine and/or Xie Ansuan, preferred L-leucine and/or L-Isoleucine and/or L-Xie Ansuan.

[0013.0.0.3] sees [0013.0.0.0]

[0014.0.3.3] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is leucine and/or Isoleucine and/or Xie Ansuan, preferred L-leucine and/or L-Isoleucine and/or L-Xie Ansuan.Therefore, in the present invention, term " fine chemicals " is meant " leucine and/or Isoleucine and/or Xie Ansuan " as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of leucine and/or Isoleucine and/or Xie Ansuan as used herein.

[0015.0.3.3] an embodiment, term " fine chemicals " meaning is meant leucine and/or Isoleucine and/or Xie Ansuan, preferred L-leucine and/or L-Isoleucine and/or L-Xie Ansuan.In whole specification sheets, term " fine chemicals " meaning is meant salt, ester or the acid amides of leucine and/or Isoleucine and/or Xie Ansuan, preferred L-leucine and/or L-Isoleucine and/or L-Xie Ansuan, its free form or is bonded to proteinic leucine and/or Isoleucine and/or Xie Ansuan, preferred L-leucine and/or L-Isoleucine and/or L-Xie Ansuan.In preferred embodiments, term " fine chemicals " meaning is meant leucine and/or Isoleucine and/or Xie Ansuan, preferred L-leucine and/or L-Isoleucine and/or L-Xie Ansuan or its salt of free form or is bonded to proteinic leucine and/or Isoleucine and/or Xie Ansuan, preferred L-leucine and/or L-Isoleucine and/or L-Xie Ansuan.

[0016.0.3.3] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327; With

(b) in allowing described biology, produce fine chemicals be leucine and/or Isoleucine and/or Xie Ansuan or comprise leucine and/or the condition of the fine chemicals of Isoleucine and/or Xie Ansuan under make biological growth.

And/or

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or have the 3rd row as Table II A or IIB, 19-29,29a-29u, the sequence of polypeptide shown in 363-385 is capable or have by as Table I A or IB the 5th or 7 row, 19-29,29a-29u, the protein of the sequence of the coded polypeptide of nucleic acid molecule shown in 363-385 is capable and

(b) in allowing described biology, produce under fine chemicals, particularly Isoleucine, Xie Ansuan and/or the leucic condition and make biological growth.

And/or

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or have by as Table II the 5th or 7 row, 19-29,29a-29u, the protein of the sequence of the coded polypeptide of nucleic acid molecule shown in 363-385 is capable and

(b) in allowing described biology, produce under the condition that fine chemicals is leucine, Xie Ansuan and/or Isoleucine or the fine chemicals that comprises leucine, Xie Ansuan and/or Isoleucine and make biological growth.

And/or

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or have by as Table II the 5th or 7 row, 19-29,29a-29u, the protein of the sequence of the coded polypeptide of nucleic acid molecule shown in 363-385 is capable and

(b) in allowing described biology, produce under the condition that fine chemicals is Xie Ansuan, leucine and/or Isoleucine or the fine chemicals that comprises Xie Ansuan, leucine and/or Isoleucine and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have as activity of proteins shown in Table II the 3rd row, 19-29,29a-29u, 363-385 are capable or have by as the sequence of the coded polypeptide of nucleic acid molecule shown in Table I the 5th or 7 row, 19-29,29a-29u, 363-385 are capable and

(b) in allowing described biology, produce under the condition of fine chemicals, particularly arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro) and make biological growth.

[0016.1.3.3] therefore, term " fine chemicals " refers to and Table I-IV 19-29 in one embodiment, 29a, 363,367,373,377,379, relevant " Isoleucine " or its homologue of the listed full sequence of 384 row, and refer in one embodiment and Table I-IV 29a-29j, 365,369-372,375,376,380,381,382, relevant " Xie Ansuan " or its homologue of the listed full sequence of 385 row, and refer in one embodiment and Table I-IV 29k-29u, 364,366,368,374,378, relevant " leucine " or its homologue of the listed full sequence of 383 row.

Therefore, in one embodiment, term " fine chemicals " refers to and Table I-IV the 363rd, 364,365,367,368 and relevant " Xie Ansuan, leucine and the Isoleucine " of the 369 listed full sequences of row, in one embodiment, term " fine chemicals " refers to and Table I-IV the 373rd and 374, relevant " leucine and the Isoleucine " of the 377 and 378 listed full sequences of row.

Therefore, term " fine chemicals " can refer to " leucine " and/or " Isoleucine " and/or " Xie Ansuan " according to environment and context.For the meaning of illustrating term " fine chemicals " is meant " arginine " and/or " L-glutamic acid " and/or " glutamine " and/or " proline(Pro) ", also can use term " each fine chemicals ".

[0017.0.0.3] sees [0017.0.0.0]

[0018.0.0.3] sees [0018.0.0.0]

The method that [0019.0.3.3] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist to as Table II the 3rd be listed as, 19-29,29a-29u, protein shown in 363-385 is capable or by as Table I the 5th or 7 row, 19-29,29a-29u, the coded activity of proteins of nucleic acid molecule shown in 363-385 is capable is carried out above-mentioned modification.

[0020.0.3.3] is surprisingly found out that, yeast saccharomyces cerevisiae protein YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042 and/or e. coli k12 protein b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 transgene expression in Arabidopis thaliana is given leucine and/or Isoleucine and/or the raising of Xie Ansuan (or fine chemicals) content of the plant that transforms.

[0021.0.0.3] sees [0021.0.0.0]

The sequence of [0022.0.3.3] yeast saccharomyces cerevisiae YDL127W has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity is defined as " G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) protein ".Therefore, in one embodiment, the inventive method comprises " G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) protein " as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of " G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) protein " is enhanced or produces, and for example " G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) protein " or the activity of its homologue from yeast saccharomyces cerevisiae is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YDR245W has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity is defined as galactosyl-(mannose group)-transferring enzyme.Therefore, in one embodiment, the inventive method comprises the purposes of galactosyl as shown here-(mannose group)-transferring enzyme or its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of galactosyl-(mannose group)-transferring enzyme is enhanced or produces, and for example is enhanced or produces from galactosyl-(the mannose group)-transferring enzyme of yeast saccharomyces cerevisiae or the activity of its homologue

The sequence of YDR271C is by Le T., Johnston M., and (in March, 1996) is committed to the EMBL/GenBank/DDBJ database, by Waterston R.; (in May, 1996) and Jia Y., (in June, 1997) is committed to the EMBL/GenBank/DDBJ database, and its cytoactive is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YDR271C from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the YDR271C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YDR271C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YER173w has been published in Dietrich, Nature 387 (6632 supplementary issue), 78-81,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity is defined as " participating in dna damage and reduction division pachytene stage check position activatory check position protein ".Therefore, in one embodiment, the inventive method comprises " participating in dna damage and reduction division pachytene stage check position activatory check position protein " as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity that " participates in dna damage and reduction division pachytene stage check position activatory check position protein " is enhanced or produces, and for example " participating in dna damage and reduction division pachytene stage check position activatory check position protein " or the activity of its homologue from yeast saccharomyces cerevisiae is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YGR101W has been published in Tettelin, H., and Nature 387 (6632 supplementary issue), 81-84,1997 and Goffeau, A., Science 274 (5287), 546-547,1996, and its activity is defined as flat rhombus proteolytic enzyme.Therefore, in one embodiment, the inventive method comprises the purposes of flat rhombus proteolytic enzyme as shown here or its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, flat rhombus protease activities is enhanced or produces, and for example is enhanced or produces from the flat rhombus proteolytic enzyme of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YJL072C has been published in Goffeau, A., and Science 274 (5287), 546-547,1996 and Galibert, F., EMBO be (9) J.15,2031-2049,1996, and its activity is defined as " subunit that chromosomal DNA duplicates required GINS complex body ".Therefore, in one embodiment, the inventive method comprises " chromosomal DNA duplicates the subunit of required GINS complex body " as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of " chromosomal DNA duplicates the subunit of required GINS complex body " is enhanced or produces, and for example " chromosomal DNA duplicates the subunit of required GINS complex body " or the activity of its homologue from yeast saccharomyces cerevisiae is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YKR057W has been published in Dujon etc., Nature 369 (6479), 371-378,1994 and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity be defined as participating in rrna biological take place and translation and to S21A, S26A and/or the similar ribosomal protein of YS25 ribosomal protein.Therefore, in one embodiment, the inventive method comprises the biological generation of the participation rrna from yeast saccharomyces cerevisiae and ribosomal protein translation and similar to S21A, S26A and/or YS25 ribosomal protein or the purposes of its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, participate in biological generation of rrna and be enhanced or produce, for example be enhanced or produce with translation and the similar ribosomal protein or the activity of its homologue to S21A, S26A and/or YS25 ribosomal protein from biological generation of the participation rrna of yeast saccharomyces cerevisiae with the activity of translation and similar ribosomal protein to S21A, S26A and/or YS25 ribosomal protein.

The sequence of yeast saccharomyces cerevisiae YNL135C has been published in Philippsen, P., Nature 387 (6632 supplementary issue), 93-98,1997 and Goffeau, A., Science 274 (5287), 546-547,1996, and its activity is defined as the peptidyl prolyl isomerase.Therefore, in one embodiment, the inventive method comprises the peptidyl prolyl isomerase as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of peptidyl prolyl isomerase is enhanced or produces, and for example is enhanced or produces from the peptidyl prolyl isomerase of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YFL013C has been published in Goffeau, A., and Science 274 (5287), 546-547,1996 and Murakami, Y., Nat.Genet.10 (3), 261-268,1995, and its activity is defined as " INO80 chromatin reconstitution complex subunit ".Therefore, in one embodiment, the inventive method comprises " INO80 chromatin reconstitution complex subunit " as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, " INO80 chromatin reconstitution complex subunit " activity of proteins is enhanced or produces, and for example is enhanced or produces from " the INO80 chromatin reconstitution complex subunit " protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YGR104C has been published in Thompson etc., Cell 73:1361-1375, and 1993, and its activity is defined as " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 ".Therefore, in one embodiment, the inventive method comprises " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 " as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 " is enhanced or produces, and for example " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 " or the activity of its homologue from yeast saccharomyces cerevisiae is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Goffeau etc., Science 274 (5287), 546-547,1996 and Churcher etc., Nature 387 (6632 supplementary issue), 84-87,1997, and its activity be defined as the S phase (DNA synthetic) initial or finish necessary chromobindins.Therefore, in one embodiment, the inventive method comprises that the S phase from yeast saccharomyces cerevisiae as shown here (DNA synthetic) is initial or finishes the purposes of necessary chromobindins or its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, activity initial or that finish necessary chromobindins was enhanced or produced the S phase (DNA synthetic), and activity for example initial from the S phase (DNA is synthetic) of yeast saccharomyces cerevisiae or that finish necessary chromobindins or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YOR350C has been published in Goffeau, A., and Science 274 (5287), 546-547,1996 and Dujon, B., Nature 387 (6632 supplementary issue), 98-102,1997, and its cytoactive is not still characterized.Therefore, in one embodiment, the inventive method comprises the YOR350C protein as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the YOR350C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YOR350C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YFR042W has been published in Goffeau etc., and Science 274 (5287), 546-547,1996 and Murakami, Y., Nat.Genet.10 (3), 261-268, its activity of 1995and is defined as " protein that the yeast cell survival is required ".Therefore, in one embodiment, the inventive method comprises " protein that the yeast cell survival is required " or the purposes of its homologue from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of " protein that the yeast cell survival is required " is enhanced or produces, and for example " protein that the yeast cell survival is required " or the activity of its homologue from yeast saccharomyces cerevisiae is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YFL019C has been published in Murakami, Y., and Nat.Genet.10 (3), 261-268,1995, and its activity is defined in the 13.7kDa protein of the supposition of PAU5-LPD1 intergenic region.Therefore, in one embodiment, the inventive method comprises the purposes by the coded protein of yeast saccharomyces cerevisiae nucleotide sequence YFL019C or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, be enhanced or produced by the coded activity of proteins of nucleotide sequence YFL019C, for example the activity of the coded protein of yeast saccharomyces cerevisiae nucleotide sequence YFL019C or its homologue is enhanced or produces.

The sequence of e. coli k12 b1708 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is defined as lipoprotein.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary lipoprotein or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of lysine decarboxylase is enhanced or produces, and for example the activity from colibacillary lysine decarboxylase or its homologue is enhanced or produces.

The sequence of e. coli k12 b1829 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is defined as having the heat shock protein (htpx) of protease activity.Therefore, in one embodiment, the inventive method comprises as shown here having the heat shock protein (htpx) of protease activity or a purposes of its homologue from colibacillary, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, activity with heat shock protein (htpx) of protease activity is enhanced or produces, and for example has the heat shock protein (htpx) of protease activity or the activity of its homologue is enhanced or produces from colibacillary.

The sequence of e. coli k12 b2957 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is defined as pericentral siphon altheine enzyme II.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary pericentral siphon altheine enzyme II or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of pericentral siphon altheine enzyme II is enhanced or produces, and for example the activity from colibacillary pericentral siphon altheine enzyme II or its homologue is enhanced or produces.

The sequence of e. coli k12 b3366 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is defined as the nitrite reductase small subunit.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary nitrite reductase small subunit or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of nitrite reductase small subunit, preferred nitrite reductase small subunit is enhanced or produces, for example from colibacillary nitrite reductase small subunit or its homologue.

The sequence of e. coli k12 b0828 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity is defined as possible asparaginase (EC:3.5.1.1) and/or ybiK protein (altheine hydroamidase).Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary possible asparaginase (EC:3.5.1.1) and/or the purposes of ybiK protein (altheine hydroamidase) or its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of possible asparaginase (EC:3.5.1.1) and/or ybiK protein (altheine hydroamidase) is enhanced or produces, and for example is enhanced or produces from the colibacillary possible asparaginase (EC:3.5.1.1) and/or the activity of ybiK protein (altheine hydroamidase) or its homologue.

The sequence of e. coli k12 b3966 has been published in R; Heller, J.Bacteriol.161,904-908,1985; R; Doublet, J.Bacteriol.174,5772-5779,1992 and R; Gustafsson, J.Bacteriol.173,1757-1764,1991, and its activity is defined as outer porin.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary outer porin or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of outer porin is enhanced or produces, and for example the activity from colibacillary outer porin or its homologue is enhanced or produces.

The sequence of e. coli k12 b4151 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is defined as fumaric reductase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary fumaric reductase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of fumaric reductase is enhanced or produces, and for example the activity from colibacillary fumaric reductase or its homologue is enhanced or produces.

The sequence of e. coli k12 b1827 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is defined as having the repressor protein (IclR family) of DNA in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprise as shown here from the colibacillary DNA of having in conjunction with the repressor protein (IclR family) in wing spirane structure territory or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, have DNA and be enhanced or produce, for example be enhanced or produce in conjunction with the repressor protein (IclR family) in wing spirane structure territory or the activity of its homologue from the colibacillary DNA of having in conjunction with the activity of the repressor protein (IclR family) in wing spirane structure territory.

The sequence of e. coli k12 b0124 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the protein with glucose dehydrogenase activity.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary Hexose phosphate dehydrogenase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of Hexose phosphate dehydrogenase is enhanced or produces, and for example the activity from colibacillary Hexose phosphate dehydrogenase or its homologue is enhanced or produces.

The sequence of e. coli k12 b0149 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the conjugated protein 1b of bi-functional penicillin g-: glycosyltransferase (N-end); Transpeptidase (C-end).Therefore, in one embodiment, the inventive method comprises as shown here from the conjugated protein 1b of colibacillary bi-functional penicillin g-: glycosyltransferase (N-end); The purposes of transpeptidase (C-end) or its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, has the conjugated protein 1b of bi-functional penicillin g-: glycosyltransferase (N-end); Transpeptidase (C-end) activity of proteins is enhanced or produces, and for example has a conjugated protein 1b of bi-functional penicillin g-from colibacillary: glycosyltransferase (N-end); The activity of transpeptidase (C-end) protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b0161 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease Do, heat shock protein; Pericentral siphon serine protease Do; Heat shock protein HtrA.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary pericentral siphon serine protease Do, heat shock protein; Pericentral siphon serine protease Do; The purposes of heat shock protein HtrA or its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, pericentral siphon serine protease Do, heat shock protein; Pericentral siphon serine protease Do; The activity of heat shock protein HtrA is enhanced or produces, for example from colibacillary pericentral siphon serine protease Do, heat shock protein; Pericentral siphon serine protease Do; The activity of heat shock protein HtrA or its homologue is enhanced or produces.

The sequence of e. coli k12 b0486 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition amino acid/amine transporter.Therefore, in one embodiment, the inventive method comprises the purposes of amino acid/amine transporter or its homologue from colibacillary supposition as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of amino acid/amine transporter of supposing is enhanced or produces, and for example is enhanced or produces from the amino acid/amine transporter of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b1313 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the desaturase with NAD (P)-binding domains and GroES structural domain.Therefore, in one embodiment, the inventive method comprises as shown here from the desaturase with NAD (P)-binding domains and GroES structural domain of colibacillary supposition or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, activity with desaturase of NAD (P)-binding domains and GroES structural domain is enhanced or produces, and for example is enhanced or produces from the desaturase of the colibacillary NAD of having (P)-binding domains and GroES structural domain or the activity of its homologue.

The sequence of e. coli k12 b1343 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be ATP RNA-dependent helicase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary ATP RNA-dependent helicase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of ATP RNA-dependent helicase is enhanced or produces, and for example the activity from colibacillary ATP RNA-dependent helicase or homologue is enhanced or produces.

The sequence of e. coli k12 b1463 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be N-hydroxy aromatic amine O-Transacetylase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary-hydroxy aromatic amine O-Transacetylase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of N-hydroxy aromatic amine O-Transacetylase is enhanced or produces, and for example the activity from colibacillary N-hydroxy aromatic amine O-Transacetylase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2022 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be bi-functional: histidinol-Phosphoric acid esterase (N-end); Imidazoles glycerol-3-phosphate dehydratase (C-end); Imidazoleglycerolphosphat dehydrase and histidinol-phosphate phosphatase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary bi-functional: histidinol-Phosphoric acid esterase (N-end); Imidazoles glycerol-3-phosphate dehydratase (C-end); The purposes of imidazoleglycerolphosphat dehydrase and histidinol-phosphate phosphatase or its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, bi-functional: histidinol-Phosphoric acid esterase (N-end); Imidazoles glycerol-3-phosphate dehydratase (C-end); The activity of imidazoles glycerol-3-phosphate dehydratase and histidinol-phosphate phosphatase is enhanced or produces, for example from colibacillary bi-functional: histidinol-Phosphoric acid esterase (N-end); Imidazoles glycerol-3-phosphate dehydratase (C-end); The activity of imidazoles glycerol-3-phosphate dehydratase and histidinol-phosphate phosphatase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2414 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit is enhanced or produces, and for example the activity from colibacillary PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue is enhanced or produces.

The sequence of e. coli k12 b2664 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be supposition have transcription repressor (GntR family) protein of DNA in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprise as shown here from colibacillary have a supposition have DNA in conjunction with transcription repressor (GntR family) the active protein in wing spirane structure territory or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the DNA that has that supposes is enhanced or produces in conjunction with the activity of the transcription repressor (GntR family) in wing spirane structure territory, and for example the DNA that has from colibacillary supposition is enhanced or produces in conjunction with the transcription repressor (GntR family) in wing spirane structure territory or the activity of its homologue.

The sequence of e. coli k12 b3117 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be catabolic PLP dependent enzyme threonine dehydra(ta)se.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary catabolic PLP dependent enzyme threonine dehydra(ta)se or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of catabolic PLP dependent enzyme threonine dehydra(ta)se is enhanced or produces, and for example the activity from colibacillary catabolic PLP dependent enzyme threonine dehydra(ta)se or its homologue is enhanced or produces.

The sequence of e. coli k12 b3256 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be acetyl-CoA carboxylase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary acetyl-CoA carboxylase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of acetyl-CoA carboxylase is enhanced or produces, and for example the activity from colibacillary acetyl-CoA carboxylase or its homologue is enhanced or produces.

The sequence of e. coli k12 b3938 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be methionine(Met) biosynthesizing transcription repressor.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary methionine(Met) biosynthesizing transcription repressor or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of methionine(Met) biosynthesizing transcription repressor is enhanced or produces, and for example the activity from colibacillary methionine(Met) biosynthesizing transcription repressor or its homologue is enhanced or produces.

The sequence of e. coli k12 b3983 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L12.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 50S ribosomal subunit protein matter L12 or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of 50S ribosomal subunit protein matter L12 is enhanced or produces, and for example the activity from colibacillary 50S ribosomal subunit protein matter L12 or its homologue is enhanced or produces.

The sequence of e. coli k12 b4054 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be tyrosine repressor type tyrosine aminotransferase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary tyrosine repressor type tyrosine aminotransferase or its homologue as shown here, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, the activity of tyrosine repressor type tyrosine aminotransferase is enhanced or produces, and for example the activity from colibacillary tyrosine repressor type tyrosine aminotransferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b4327 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the HTH-type transcriptional (LysR family) with periplasmic binding protein structural domain.Therefore, in one embodiment, the inventive method comprises as shown here having the HTH-type transcriptional (LysR family) of periplasmic binding protein structural domain or a purposes of its homologue from colibacillary, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, activity with HTH-type transcriptional (LysR family) of periplasmic binding protein structural domain is enhanced or produces, and for example has the HTH-type transcriptional (LysR family) of periplasmic binding protein structural domain or the activity of its homologue is enhanced or produces from colibacillary.

[0023.0.3.3] in one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W and/or YFL019C is to have described activity and from Eukaryotic homologue.In one embodiment, the homologue of b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 is to have described activity and from the homologue of bacterium.In one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is to have described activity and from the homologue of fungi.In one embodiment, the homologue of b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 is to have described activity and from the homologue of Proteobacteria.In one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is to have described activity and from the homologue of Ascomycota.In one embodiment, the homologue of b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 is to have described activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is to have described activity and from saccharomycetic homologue.In one embodiment, the homologue of b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 is to have described activity and from enterobacteria purpose homologue.In one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is to have described activity and from the homologue of yeast guiding principle.In one embodiment, the homologue of b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 is to have described activity and from the homologue of enterobacteriaceae.In one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is to have described activity and from the homologue of Saccharomycetes.In one embodiment, the homologue of b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 is to have described activity and from the homologue of Escherichia.In one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is to have described activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is to have described activity and from the homologue of yeast guiding principle.

The homologue of polypeptide shown in [0023.1.0.3] Table II the 3rd row, 19-29,29a-29u, 363-385 are capable can be by Table I the 7th row, be respectively 19-29,29a-29u, 363-385 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, be respectively 19-29,29a-29u, 363-385 capable shown in polypeptide.

[0024.0.0.3] other homologue is described in hereinafter.

[0025.0.3.3] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause biology or its part, increase of leucine and/or Isoleucine and/or Xie Ansuan level and protein have YDL127W in the preferred described biomass cells, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the proteinic above-mentioned activity of b4054 and/or b4327, then described protein or polypeptide have YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 activity of proteins.In this manual, if this kind protein or polypeptide still have YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL 019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, proteinic biology of b4054 and/or b4327 or the enzyme activity, if promptly with yeast saccharomyces cerevisiae YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W and/or e. coli k12 b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein are compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, the most preferred 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.3] in one embodiment, if it comes comfortable the evolution to go up biology with the biological apart from each other of expressing it, the used polypeptide of polypeptide of the present invention or the inventive method is still given described activity, as increase fine chemicals in biological or its part.For example play eozoan and express biology from different sections, order, guiding principle or door.

[0025.2.0.3] in one embodiment, if polypeptide come comfortable evolve go up with biophase shown in Table I the 4th row apart from nearer biological and in evolution with in the biology of initial biological apart from each other, expressing, polypeptide of the present invention or the polypeptide that is used for the inventive method can be given described activity, as increase fine chemicals in biological or its part.For example just eozoan is biological from different sections, order, guiding principle or door with expression, and biology comes from identical section, order, guiding principle or door shown in first eozoan and Table I the 4th row.

[0026.0.0.3] to [0033.0.0.3]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.3.3] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention, for example, it has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein active, or by Table I the 5th row, 19-29,29a-29u, the protein of nucleic acid molecule encoding shown in 363-385 is capable or its homologue are (as Table I the 7th row, 19-29,29a-29u, homologue shown in 363-385 is capable) expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and shows the fine chemicals quantity that increases.

[0035.0.0.3] to [0044.0.0.3]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.3.3] is at yeast saccharomyces cerevisiae protein YDL127W or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 19th row or participate under the situation that the activity of " G1/S-specific cell cyclin PCL2 (the cyclin HCS26 homologue) protein " of mitotic cell cycle and cell cycle control is enhanced, preferably, give fine chemicals, preferred Isoleucine increase by 33% or higher in one embodiment.

At yeast saccharomyces cerevisiae protein YDR245W or its homologue, for example Table I A or IB the 5th or 7 row, 29k capable shown in or participate in the C-compound with the carbohydrate utilization, sprout, the cell polarization and becomes thread, under the situation that the activity of galactosyl-(the mannose group)-transferring enzyme of protein modification is enhanced, preferably, give in one embodiment fine chemicals, preferred leucine be increased in 50% and 173% or higher between.

Under the situation that the activity of yeast saccharomyces cerevisiae protein YDR271C or its homologue is enhanced, preferably, give in one embodiment fine chemicals, preferred Isoleucine be increased in 30% and 74% or higher between.

At yeast saccharomyces cerevisiae protein YER173w or its homologue, for example Table I A or IB the 5th or 7 row, 29b capable shown in or participate in dna damage and reduction division pachytene stage check position activatory check position protein; DNA reorganization and DNA repair, cell cycle check position (check position of form generation, dna damage, dna replication dna, mitotic division phase and spindle body), under the situation that nucleic acid activity synthetic in conjunction with, DNA and that duplicate is enhanced, preferably, give in one embodiment fine chemicals be increased in 18% and 187% between, preferred Xie Ansuan be increased in 18% and 82% between or leucine be increased in 93% and 187% or higher between.

At yeast saccharomyces cerevisiae protein YGR101W or its homologue, for example Table I A or IB the 5th or 7 row, 29c capable shown in or participate under the situation that the flat rhombus protease activities of cell communication/signal transduction mechanism is enhanced, preferably, in one embodiment, give fine chemicals, preferred Xie Ansuan be increased in 47% and 60% or higher between.

At yeast saccharomyces cerevisiae protein YJL072C or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 21st row or participate in that cytodifferentiation, late embryo are grown, system regulates/grow under the situation that " chromosomal DNA duplicates the subunit of the required GINS complex body " activity of proteins of (form generation) is enhanced with environmental interaction, directivity cell, preferably, give in one embodiment fine chemicals, preferred Isoleucine be increased in 31% and 356% or higher between.

At yeast saccharomyces cerevisiae protein YKR057W or its homologue, for example Table I A or IB the 5th or 7 row, 29d capable shown in or participate under the situation that the activity of the biological generation of rrna, cytodifferentiation and ribosomal protein translation and similar to S21A, S26A and/or YS25 ribosomal protein is enhanced, preferably, give in one embodiment fine chemicals, preferred Xie Ansuan be increased in 19% and 100% or higher between.

At yeast saccharomyces cerevisiae protein YNL135C or its homologue, for example Table I A or IB the 5th or 7 row, 29m capable shown in or participate under the situation that the activity of the peptidyl prolyl isomerase of protein folding and stability, protein target, sorting and transposition, protein synthesis is enhanced, preferably, give in one embodiment fine chemicals, preferred leucine be increased in 45% and 280% or higher between.

At yeast saccharomyces cerevisiae protein YFL013C or its homologue, for example Table I A or IB the 5th or 7 row, 29e capable shown in or under the activity of " the INO80 chromatin reconstitution complex subunit " situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred Xie Ansuan be increased in 22% and 58% or higher between.

At yeast saccharomyces cerevisiae protein YGR104C or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 22nd row or participate under the situation that the activity of " yeast rna polymerase II repressor SRB5 and/or the RNA polymerase B repressor SRB5 " transcribe is enhanced, preferably, give in one embodiment fine chemicals, preferred Isoleucine be increased in 33% and 102% or higher between.

At yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 23rd row or participate in DNA synthetic and duplicate, S phase (DNA is synthetic) of mitotic cell cycle and cell cycle control is initial or finish under the situation that the activity of necessary chromobindins is enhanced, preferably, give in one embodiment fine chemicals be increased in 18% and 535% between, preferred Xie Ansuan be increased in 18% and 314% between or Isoleucine increase by 535%.

Under the situation that the activity of yeast saccharomyces cerevisiae protein YOR350C or its homologue is enhanced, preferably, give in one embodiment fine chemicals be increased in 101% and 348% between, preferred leucine be increased in 247% and 348% between or Isoleucine be increased between 101% and 178%.

At yeast saccharomyces cerevisiae protein YFR042W or its homologue, for example Table I A or IB the 5th or 7 row, 29n capable shown in or under the activity of " the yeast cell survival required protein " situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred leucine is increased between 43% and 325%.

At yeast saccharomyces cerevisiae protein YFL019C or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 385th row or under the situation that the 13.7kDa activity of proteins of the supposition of PAU5-LPD1 intergenic region is enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 22% and 58%.

At e. coli k12 protein b1708 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 25th row or participate in that cell growth/form takes place, division of cytoplasm (cell fission)/under the situation that the activity of the lipoprotein of formation and sporulation is enhanced, preferably, in one embodiment, give fine chemicals be increased in 33% and 333% between, preferred Xie Ansuan be increased in 33% and 149% between, Isoleucine be increased in 51% and 290% between and leucine be increased between 60% and 333%.

Activity at e. coli k12 protein b1829 or its homologue is enhanced, shown in for example Table I A or IB the 5th or 7 row, the 26th row or participate in coercing under the situation that the activity of the heat shock protein with protease activity (htpx) of reaction, pheromone reaction, mating type decision, protein modification, proteolytic degradation is enhanced, preferably, give in one embodiment fine chemicals be increased in 16% and 1480% between, preferred Isoleucine be increased in 77% and 1480% between and bright ammonia be increased between 60% and 1200%.

At e. coli k12 protein b2957 or its homologue, for example Table I A or IB the 5th or 7 row, 29h capable shown in or participate under the situation that the activity of the pericentral siphon altheine enzyme II of aspartic acid biosynthesizing, l-asparagine biosynthesizing, nitrogen and sulphur utilization, amino acid degradation (katabolism) is enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 32% and 303%.

At e. coli k12 protein b3366 or its homologue, for example Table I A or IB the 5th or 7 row, 29r capable shown in or under the activity of the nitrite reductase small subunit situation about being enhanced, preferably, give in one embodiment fine chemicals be increased in 39% and 581% between, preferred Isoleucine be increased in 39% and 327% between and leucine be increased between 65% and 581%.

At e. coli k12 protein b0828 or its homologue, for example Table I A or IB the 5th or 7 are listed as, shown in the 28th row or participate in amino acid degradation (katabolism), proteolytic degradation, protein synthesis, aspartic acid family amino acid degradation, under the situation that the activity of biosynthetic asparaginase of glucosides (EC:3.5.1.1) and/or ybiK protein (altheine hydroamidase) is enhanced, preferably, giving fine chemicals in one embodiment is increased between 20% and 102%, between preferred Xie Ansuan is increased in 20% and 61% and Isoleucine be increased between 60% and 102%.

At e. coli k12 protein b3966 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 29th row or under the activity of the outer porin situation about being enhanced, preferably, give in one embodiment fine chemicals be increased in 59% and 647% between, preferred Isoleucine be increased in 79% and 392% between and leucine be increased between 59% and 647%.

At e. coli k12 protein b4151 or its homologue, for example Table I A or IB the 5th or 7 row, 29t capable shown in or participate in transcribing the having under the situation that DNA is enhanced in conjunction with the activity of the repressor protein (IclR family) in wing spirane structure territory of control, preferably, give fine chemicals in one embodiment, preferred leucine is increased between 31% and 46%.

At e. coli k12 protein b1827 or its homologue, for example Table I A or IB the 5th or 7 row, 29a capable shown in or under the activity of the fumaric reductase situation about being enhanced, preferably, give in one embodiment fine chemicals be increased in 24% and 884% between, preferred Xie Ansuan be increased in 24% and 192% between, Isoleucine be increased in 81% and 582% between and leucine be increased between 63% and 884%.

At e. coli k12 protein b0124 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 363rd row or under the activity of the Hexose phosphate dehydrogenase situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred Isoleucine be increased in 43% and 638% between, preferred leucine be increased in 56% and 570% between, preferred Xie Ansuan is increased between 24% and 268%.

At e. coli k12 protein b0149 or its homologue, for example Table I A or IB the 5th or 7 row, the 366th the row shown in or the conjugated protein 1b of bi-functional penicillin g-: glycosyltransferase (N-end); Under the situation that the activity of transpeptidase (C-end) is enhanced, preferably, give fine chemicals in one embodiment, preferred leucine is increased between 62% and 99%.

At e. coli k12 protein b0161 or its homologue, for example Table I A or IB the 5th or 7 row, the 367th the row shown in or the pericentral siphon serine protease, under the situation that the activity of heat shock protein is enhanced, preferably, give in one embodiment fine chemicals, preferred leucine be increased in 71% and 1140% between, preferred Xie Ansuan be increased in 29% and 399% between, preferred Isoleucine is increased between 98% and 1030%.

At e. coli k12 protein b0486 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 370th row or under the situation that amino acid/the amine transporter activity is enhanced of supposition, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 22% and 181%.

At e. coli k12 protein b1313 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 371st row or have under the situation that the activity of desaturase of the supposition of NAD (P)-binding domains and GroES structural domain is enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 22% and 50%.

At e. coli k12 protein b1343 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 372nd row or under the activity of the ATP RNA-dependent helicase situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 21% and 23%.

At e. coli k12 protein b1463 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 373rd row or under the activity of the N-hydroxy aromatic amine O-Transacetylase situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred Isoleucine be increased in 39% and 119% between, preferred leucine is increased between 66% and 157%.

At e. coli k12 protein b2022 or its homologue, for example Table I A or IB the 5th or 7 row, the 375th the row shown in or bi-functional: histidinol-Phosphoric acid esterase (N-end); Imidazoles glycerol-3-phosphate dehydratase (C-end); Under the situation that the activity of imidazoleglycerolphosphat dehydrase and histidinol-phosphate phosphatase is enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 22% and 26%.

At e. coli k12 protein b2414 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 376th row or under the activity of PLP dependent enzyme cysteine synthase A and the O-acetylserine sulfhydrylase A subunit situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 35% and 139%.

At e. coli k12 protein b2664 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 377th row or the having under the situation that DNA is enhanced in conjunction with the activity of the transcription repressor (GntR family) in wing spirane structure territory of supposition, preferably, give in one embodiment fine chemicals, preferred leucine be increased in 67% and 1050% between, preferred Isoleucine is increased between 42% and 1170%.

At e. coli k12 protein b3117 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 379th row or under the activity of the catabolic PLP dependent enzyme threonine dehydra(ta)se situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred Isoleucine is increased between 47% and 656%.

At e. coli k12 protein b3256 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 380th row or under the activity of the acetyl-CoA carboxylase situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 27% and 36%.

At e. coli k12 protein b3938 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 381st row or under the activity of the methionine(Met) biosynthesizing transcription repressor situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 24% and 42%.

At e. coli k12 protein b3983 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 382nd row or under the activity of the 50S ribosomal subunit protein matter L12 situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred Xie Ansuan is increased between 24% and 202%.

At e. coli k12 protein b4054 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 383rd row or under the activity of the tyrosine repressor type tyrosine aminotransferase situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred leucine is increased between 126% and 163%.

At e. coli k12 protein b4327 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 384th row or have under the situation that the activity of the HTH-type transcriptional (LysR family) of periplasmic binding protein structural domain is enhanced, preferably, give being increased between 42% and 54% of fine chemicals, preferred Isoleucine in one embodiment.

[0046.0.3.3] is at yeast saccharomyces cerevisiae protein YDL127W YER173w or its homologue, for example under the situation that " G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) protein " check position activity of proteins is enhanced, preferably giving fine chemicals and tryptophane increases.

Under the situation that the activity of yeast saccharomyces cerevisiae protein YDR271C or its homologue is enhanced, preferably giving fine chemicals and proline(Pro) increases.

At yeast saccharomyces cerevisiae protein YER173w or its homologue, for example under the situation that the check position activity of proteins is enhanced, preferably giving fine chemicals and tryptophane increases.

At yeast saccharomyces cerevisiae protein YGR101W or its homologue, under the situation that for example flat rhombus protease activities is enhanced, preferably giving fine chemicals and phenylalanine increases.

At yeast saccharomyces cerevisiae protein YJL072C or its homologue, for example under the situation that " chromosomal DNA duplicates the subunit of required GINS complex body " activity of proteins is enhanced, preferably giving fine chemicals and phenylalanine increases.

At yeast saccharomyces cerevisiae protein YKR057W or its homologue, under the situation that the activity of for example similar to S21A, S26A and/or YS25 ribosomal protein ribosomal protein is enhanced, preferably giving fine chemicals and Threonine increases.

At yeast saccharomyces cerevisiae protein YFL013C or its homologue, for example under the situation that the activity of " INO80 chromatin reconstitution complex subunit " is enhanced, preferably give fine chemicals and 2,3-dimethyl-5-phytyl quinone increases.

At yeast saccharomyces cerevisiae protein YGR104C or its homologue, for example under the situation that the activity of " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 " rna plymerase ii repressor (yeast SRB5) is enhanced, preferably giving fine chemicals and L-glutamic acid increases.

At yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example participate in DNA synthetic and duplicate, S phase (DNA is synthetic) of mitotic cell cycle and cell cycle control is initial or finish under the situation that the activity of necessary chromobindins is enhanced, preferably giving fine chemicals and Threonine increases.

Under the situation that the activity of yeast saccharomyces cerevisiae protein YOR350C or its homologue is enhanced, preferably giving fine chemicals and tyrosine increases.

At yeast saccharomyces cerevisiae protein YFR042W or its homologue, for example under the situation that the activity of " protein that the yeast cell survival is required " is enhanced, preferably giving fine chemicals and glutamine increases.

At e. coli k12 protein b1708 or its homologue, for example participate in that cell growth/form takes place, division of cytoplasm (cell fission)/every form and situation that the activity of the lipoprotein of sporulation is enhanced under, preferably giving fine chemicals and phenylalanine increases.

Activity at e. coli k12 protein b1829 or its homologue is enhanced, and for example has under the situation that the activity of the heat shock protein (htpx) of protease activity is enhanced, and preferably giving fine chemicals and Threonine increases.

At e. coli k12 protein b3966 or its homologue, for example under the situation that the activity of outer porin is enhanced, preferably giving fine chemicals and Threonine increases.

At e. coli k12 protein b1827 or its homologue, for example under the situation that the activity of fumaric reductase is enhanced, preferably giving fine chemicals and proline(Pro) increases.

At yeast saccharomyces cerevisiae protein YFL019C or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 385th row or under the situation that the 13.7kDa activity of proteins of the supposition of PAU5-LPD1 intergenic region is enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b0124 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 363rd row or under the activity of the Hexose phosphate dehydrogenase situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b0149 or its homologue, for example Table I A or IB the 5th or 7 row, the 366th the row shown in or the conjugated protein 1b of bi-functional penicillin g-: glycosyltransferase (N-end); Under the situation that the activity of transpeptidase (C-end) is enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b0161 or its homologue, for example Table I A or IB the 5th or 7 row, the 367th the row shown in or the pericentral siphon serine protease, under the situation that the activity of heat shock protein is enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b0486 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 370th row or under the activity of amino acid/amine transporter of the supposition situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b1313 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 371st row or have under the situation that the activity of desaturase of the supposition of NAD (P)-binding domains and GroES structural domain is enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b1343 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 372nd row or under the activity of the ATP RNA-dependent helicase situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b1463 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 373rd row or under the activity of the N-hydroxy aromatic amine O-Transacetylase situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b2022 or its homologue, for example Table I A or IB the 5th or 7 row, the 375th the row shown in or bi-functional: histidinol-Phosphoric acid esterase (N-end); Under the situation that the activity of imidazoles glycerol-3-phosphate dehydratase is enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b2414 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 376th row or under the activity of PLP dependent enzyme cysteine synthase A and the O-acetylserine sulfhydrylase A subunit situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b2664 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 377th row or the having under the situation that DNA is enhanced in conjunction with the activity of the transcription repressor (GntR family) in wing spirane structure territory of supposition, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b3117 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 379th row or under the activity of the catabolic PLP dependent enzyme threonine dehydra(ta)se situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b3256 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 380th row or under the activity of the acetyl-CoA carboxylase situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b3938 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 381st row or under the activity of the methionine(Met) biosynthesizing transcription repressor situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b3983 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 382nd row or under the activity of the 50S ribosomal subunit protein matter L12 situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b4054 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 383rd row or under the activity of the tyrosine repressor type tyrosine aminotransferase situation about being enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

At e. coli k12 protein b4327 or its homologue, for example shown in Table I A or IB the 5th or 7 row, the 384th row or have under the situation that the activity of the HTH-type transcriptional (LysR family) of periplasmic binding protein structural domain is enhanced, preferably, giving the fine chemicals of free or protein bound form and other amino acid in one embodiment increases.

[0047.0.0.3] sees [0047.0.0.0]

[0048.0.0.3] sees [0048.0.0.0]

[0049.0.3.3] has to give and improves fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, 19-29,29a-29u, the polypeptide of consensus sequence shown in 363-385 is capable, perhaps as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, its function homologue shown in 363-385 is capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, 19-29,29a-29u, nucleic acid molecule shown in 363-385 is capable or its function homologue as herein described) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.3.3] for the purposes of the present invention, term " leucine " and/or " Isoleucine " and/or " Xie Ansuan " and " L-leucine " and/or " the L-Isoleucine " and/or " L-Xie Ansuan " also comprise corresponding salt, for example vitriol of the hydrochloride of leucine and/or Isoleucine and/or Xie Ansuan or leucine and/or Isoleucine and/or Xie Ansuan.Preferably, term leucine and/or Isoleucine and/or Xie Ansuan are intended to comprise term L-leucine and/or L-Isoleucine and/or L-Xie Ansuan.

[0051.0.0.3] sees [0051.0.0.0]

[0052.0.0.3] sees [0052.0.0.0]

[0053.0.3.3] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention (for example Table II the 5th and 7 row, 19-29,29a-29u, has a YDL127W shown in 363-385 is capable, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue) express increase, have the activity of the raising leucine described in the literary composition and/or Isoleucine and/or Xie Ansuan;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention and (are for example had YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue) express to improve or mRNA that coding has an active polypeptide of the present invention of the raising leucine described in the literary composition and/or Isoleucine and/or Xie Ansuan expresses and improves;

(c) improve the protein specific activity, described protein is given the active of the raising leucine that has described in the literary composition and/or Isoleucine and/or Xie Ansuan, protein or the of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein is given the active of the raising leucine that has described in the literary composition and/or Isoleucine and/or Xie Ansuan, by the coded protein of nucleic acid molecule of the present invention, polypeptide perhaps of the present invention (for example has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue) express and improve;

(e) by adding the activity that one or more external source inducible factors come stimulating protein in biological or its part, described protein is given the active of the raising leucine that has described in the literary composition and/or Isoleucine and/or Xie Ansuan, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue) express and improve;

(f) express the transgenosis of coded protein, described protein is given the raising leucine that has described in the literary composition and/or the activity of Isoleucine and/or Xie Ansuan, (for example has YDL127W by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue) express and improve; And/or

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, and described nucleic acid molecule encoding has the active of the raising leucine described in the literary composition and/or Isoleucine and/or Xie Ansuan, (for example has YDL127W by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue);

(h) by adding positive Expression element or removing negative Expression element and improve code book invention polypeptide and (for example have YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or the active polypeptide of its homologue) the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or remove straining element from control region.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system; And/or

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces;

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.2.3] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, (for example Table II the 5th and 7 is listed as for b4054 and/or b4327 protein or its homologue, 19-29,29a-29u, shown in 363-385 is capable) give leucine and/or Isoleucine and/or Xie Ansuan after active expression of polypeptides or the activity and increase.

[0055.0.0.3] to [0064.0.0.3] sees that [0055.0.0.0] is to [0064.0.0.0]

[0065.0.3.3] also can improve and have above-mentioned activity and (for example have YDL127W by introducing synthetic transcription factor, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein active) the activation of endogenous polypeptide, the activation of polypeptide of the present invention (for example giving leucine and/or Isoleucine and/or Xie Ansuan after improving expression or activity increases), described synthetic transcription factor and YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, combine closely and activate it and transcribe in the coding region of b4054 and/or b4327 protein coding gene.Can resolve the chimeric zinc finger protein that contains specific DNA binding domains and activation structure territory (for example VP16 structural domain of hsv).Specific binding domains can be in conjunction with YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the regulatory region of b4054 and/or b4327 protein coding gene.The expression of chimeric transcription factor in biological (particularly plant) causes YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the proteinic specifically expressing of b4054 and/or b4327, see for example WO01/52620, Oriz, Proc.Natl.Acad.Sci.USA, 2002, the 99th volume, 13290 or Guan, Proc.Natl.Acad.Sci.USA, 2002, the 99th volume, 13296.

[0066.0.0.3] to [0069.0.0.3]: see that [0066.0.0.0] is to [0069.0.0.0]

[0070.0.3.3] since will give nucleic acid molecule of the present invention or expression of polypeptides of the present invention a gene or a plurality of gene (nucleic acid construct of mentioning for example) or the coding YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the proteinic gene of b4054 and/or b4327 or a plurality of gene import biology separately or with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example contain the tryptophane of high level (from the physiology of nutrition angle), methionine(Met), the favorable amino acid composition of Methionin and/or Threonine.

[0071.0.0.3] sees [0071.0.0.0]

[0072.0.3.3] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has the 2-acetylactis except leucine and/or Isoleucine and/or Xie Ansuan; 2-3-dihydroxyl isovaleric acid; 2-oxo isovaleric acid; the 3-hydroxy-iso-butyric acid; 3-hydroxyl-isobutyryl-CoA; methyl acrylyl-CoA; isobutyryl-CoA; 2-ethanoyl-2-hydroxybutyric acid; 2,3-dihydroxyl-3 methylvaleric acid; 2-oxo-3 methylvaleric acid; 2-methylacetoacetyl-CoA; 2-methyl-3-maloyl group-CoA; tiglyl-CoA; 2-methylbutyryl-CoA; the 22-isopropylmolic acid; the 3-isopropylmolic acid; the oxo leucine; isovaleryl-CoA; 3-tiglyl-CoA and/or 3-methylpentene two acyls-CoA.

[0073.0.3.3] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 activity of proteins or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention are for example given biology (preferred microorganism, the non-human animal, plant or zooblast, plant or animal tissues or plant) in fine chemicals improve;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or bonded fine chemicals by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation, and optional other dissociates and/or in conjunction with amino acid.

[0074.0.0.3] to [0084.0.0.3]: see that [0074.0.0.0] is to [0084.0.0.0]

[0085.0.3.3] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th and 7 row, 19-29,29a-29u, 363-385 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th and 7 row, 19-29,29a-29u, 363-385 are capable, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.3]: see [0086.0.0.0]

[0087.0.0.3]: see [0087.0.0.0]

[0088.0.3.3] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified aminoacids content.Often be subject to minority indispensable amino acid (as Methionin, Threonine or methionine(Met) or tryptophane) owing to for example be used to raise the nutritive value of the plant of monogastric animal, so this is very important for the plant breeder.Improving or producing YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, after b4054 and/or the b4327 activity of proteins, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, the transgenic plant that produced grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0089.0.0.3] to [0097.0.0.3]: see that [0089.0.0.0] is to [0097.0.0.0]

[0098.0.3.3] in preferred embodiments, fine chemicals (leucine and/or Isoleucine and/or Xie Ansuan) be produce according to the present invention and carry out isolating where necessary.The aminoacid mixture that the method according to this invention produces other amino acid such as methionine(Met), Methionin and/or Threonine is favourable.

[0099.0.0.3] to [0102.0.0.3]: see that [0099.0.0.0] is to [0102.0.0.0]

[0103.0.3.3] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferred encoding mature form at least as polypeptide or its segmental nucleic acid molecule shown in Table II A or IIB the 5th and 7 row, 19-29,29a-29u, 363-385 are capable, it is given, and fine chemicals quantity increases in biology or its part;

(b) preferably comprise at least mature form, as the nucleic acid molecule of nucleic acid molecule shown in Table I A or IB the 5th and 7 row, 19-29,29a-29u, 363-385 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(h) nucleic acid molecule, it comprises by the nucleic acid molecule that uses Table III the 7th row, 19-29,29a-29u, the 363-385 primer in capable that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives the increase of fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise Table IV the 7th row, 19-29,29a-29u, 363-385 capable shown in consensus sequence and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding schedule IA or IB the 5th and 7 row, 19-29,29a-29u, 363-385 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0104.0.3.3] in one embodiment, sequence shown in the inventive method nucleic acid molecule used therefor and Table I A or IB the 5th and 7 row, 19-29,29a-29u, 363-385 are capable, preferably show LA the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in the sequence difference be one or more Nucleotide, perhaps the used nucleic acid of the inventive method not by Table I A or IB the 5th and 7 row, 19-29,29a-29u, 363-385 capable shown in sequence, preferred Table I A the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I A or IB 19-29,29a-29u, 363-385 capable shown in sequence, preferred Table I A the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule HA or IIB 19-29,29a-29u, 363-385 capable shown in sequence, preferred Table I A the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in polypeptide of sequence.

[0105.0.0.3] to [0107.0.0.3]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.3.3] method of the present invention and had as Table I A or IB 19-29,29a-29u, the nucleic acid molecule of sequence shown in 363-385 is capable, from being listed as Table II A or IIB the 5th or 7,19-29,29a-29u, aminoacid sequence shown in 363-385 is capable is derived or is listed as from containing just like Table IV the 7th, 19-29,29a-29u, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 363-385 is capable, perhaps its coding has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, give derivative or the homologue that leucine and/or Isoleucine and/or Xie Ansuan increase behind proteinic enzymic activity of b4054 and/or b4327 or bioactive polypeptide or its expression or active the increasing.

[0109.0.0.3] sees [0109.0.0.0]

[0110.0.0.3] helps the inventive method and coding has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the nucleic acid molecule of the polypeptide of b4054 and/or b4327 protein active can be determined from generally open database.

[0111.0.0.3] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.3.3] the inventive method is the isolated nucleic acid sequences form, its coding has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the polypeptide of b4054 and/or b4327 protein active, and give leucine and/or Isoleucine and/or Xie Ansuan increase.

[0113.0.0.3] to [0120.0.0.3]: see that [0113.0.0.0] is to [0120.0.0.0]

[0121.0.3.3] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with Table II A or IIB the 5th or 7 row, 19-29,29a-29u, peptide sequence shown in 363-385 is capable or its functional homologue as herein described have the difference of one or more amino acid moleculars, described artificial sequence is preferably given aforementioned activity, promptly giving leucine and/or Isoleucine and/or Xie Ansuan after improving its activity increases, perhaps has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 activity of proteins.

[0122.0.0.3] to [127.0.0.3]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.3.3] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 19-29,29a-29u, 363-385 are capable shown in) can be based on sequence shown in this paper, as the sequence shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable or from producing as sequence deutero-sequence shown in Table II A or IIB 19-29,29a-29u, 363-385 are capable.

[0129.0.3.3] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of the inventive method (the particularly sequence of polypeptide of the present invention).Indicate in the shown in the drawings comparison of the conservative region of polypeptide of the present invention.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence was from described comparison shown in Table IV the 7th row, 19-29,29a-29u, 363-385 were capable.

[0130.0.3.3] can use degenerated primer to have aforementioned activity (as improving expression or active back or having YDL127W by pcr amplification then, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, giving leucine and/or Isoleucine and/or Xie Ansuan after the activity of b4054 and/or b4327 increases) the new protein fragment or derive from other functional homologue of the polypeptide of the present invention of other biological.

[0131.0.0.3] to [0138.0.0.3]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.3.3] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving fine chemicals increases), described dna sequence dna under loose hybridization conditions with Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in sequence hybridization, and coding is expressed and is had the leucine of increasing and/or Isoleucine and/or the active peptide of Xie Ansuan.

[0140.0.0.3] to [0146.0.0.3]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.3.3] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it with Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, excellent Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 capable shown in one of nucleotide sequence fully complementary, thereby one of nucleotide sequence shown in can be with Table I A or IB 19-29,29a-29u, 363-385 capable is hybridized and is formed stable duplex.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.3.3] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 is capable, preferred Table I B the 7th row, 19-29,29a-29u, nucleotide sequence shown in 363-385 is capable or its portion homologous are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have and improving active or improving YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, increase the activity of leucine and/or Isoleucine and/or Xie Ansuan after the activity of b4054 and/or b4327 gene product.

[0149.0.3.3] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 is capable, preferred Table I B the 7th row, 19-29,29a-29u, one of nucleotide sequence shown in 363-385 is capable or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding has the protein of above-mentioned activity (as giving leucine and/or Isoleucine and/or Xie Ansuan increase) and randomly has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the activity of b4054 and/or b4327.

[0150.0.3.3] in addition, nucleic acid molecule of the present invention can only contain Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving leucine and/or Isoleucine and/or Xie Ansuan when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in sense strand, Table I A or IB 19-29,29a-29u, the 363-385 of one of sequence capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 19-29,29a-29u, the PCR of primer will produce YDL127W shown in 363-385 is capable, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the fragment of b4054 and/or b4327 gene product.

[0151.0.0.3] sees [0151.0.0.0]

[0152.0.3.3] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as the abundant homology of aminoacid sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable, make this albumen or its part keep to participate in produce the ability of fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising leucine as be shown in the examples and/or Isoleucine and/or Xie Ansuan.

[0153.0.3.3] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing fine chemicals.For example has YDL127W as herein described, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the activity of b4054 and/or b4327.

[0154.0.3.3] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving fine chemicals increases.

[0155.0.0.3] and [0156.0.0.3]: see [0155.0.0.0] and [0156.0.0.0]

[0157.0.3.3] the present invention relate in addition owing to the genetic code degeneracy be different from Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology fine chemicals increase), for example by Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in the polypeptide or the functional homologous compound of sequence encoding.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as the sequence shown in Table I the 5th or 7 row, capable, the preferred Table I A the 5th of 19-29,29a-29u, 363-385 or 7 row, 19-29,29a-29u, 363-385 are capable.Preferably, nucleic acid molecule of the present invention is a functional homologous compound or identical with nucleic acid molecule shown in Table I B the 7th row, 19-29,29a-29u, 363-385 are capable.

[0158.0.0.3] to [0160.0.0.3]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.3.3] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.3]: see [0162.0.0.0]

[0163.0.3.3] preferably, under stringent condition with Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.3]: see [0164.0.0.0]

[0165.0.3.3] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in sequence).

[0166.0.0.3] and [0167.0.0.3]: see [0166.0.0.0] and [0167.0.0.0]

[0168.0.3.3] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.Sequence contained during these amino acid sequence of polypeptide and Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable is different, but has kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the capable aminoacid sequence of Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of fine chemicals.Preferably, the protein of this nucleic acid molecule encoding and Table II A or IIB the 5th or 7 row, 19-29,29a-29u, the sequence that 363-385 is capable is identical at least about 60%, more preferably with Table II A or IIB the 5th or 7 row, 19-29,29a-29u, one of sequence that 363-385 is capable is identical at least about 70%, even more preferably be listed as with Table II A or IIB the 5th or 7,19-29,29a-29u, the capable sequence of 363-385 is at least about 80%, 90% or 95% homology, and most preferably be listed as with Table II A or IIB the 5th or 7,19-29,29a-29u, the capable sequence of 363-385 is at least about 96%, 97%, 98% or 99% is identical.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are different with sequence contained in sequence shown in Table II the 5th or 7 row, capable, preferred Table II B the 7th row of 19-29,29a-29u, 363-385,19-29,29a-29u, 363-385 are capable, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as aminoacid sequence shown in Table II the 5th or 7 row, capable, preferred Table II B the 7th row of 19-29,29a-29u, 363-385,19-29,29a-29u, 363-385 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in the generation that each increases fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 19-29,29a-29u, 363-385 is capable, preferred Table II B the 7th row, 19-29,29a-29u, sequence was identical at least about 60% shown in 363-385 was capable, more preferably with as Table II the 5th or 7 be listed as, 19-29,29a-29u, 363-385 is capable, preferred Table II B the 7th row, 19-29,29a-29u, one of sequence was identical at least about 70% shown in 363-385 was capable, even more preferably with as Table II the 5th or 7 be listed as, 19-29,29a-29u, 363-385 is capable, preferred Table II B the 7th row, 19-29,29a-29u, sequence was at least about 80% shown in 363-385 was capable, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 19-29,29a-29u, 363-385 is capable, preferred Table II B the 7th row, 19-29,29a-29u, sequence was at least about 96% shown in 363-385 was capable, 97%, 98% or 99% is identical.

[0169.0.0.3] to [0172.0.0.3]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.3.3] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:899 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:899 sequence before use.

[0174.0.0.3]: see [0174.0.0.0]

[0175.0.3.3] for example, the sequence that has 80% homology at protein level and SEQ ID NO:900 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQID NO:900 sequence.

[0176.0.3.3] is by replacing, insert or lack from being listed as Table II A or IIB the 5th or 7 according to of the present invention, 19-29,29a-29u, the functional equivalent that one of polypeptide obtained shown in 363-385 was capable be listed as according to of the present invention as Table II A or IIB the 5th or 7,19-29,29a-29u, one of polypeptide had at least 30% shown in 363-385 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, polypeptide shown in 363-385 is capable has essentially identical character and discerns.

[0177.0.3.3] is by replacing, insert or lack from being listed as Table I A or IB the 5th or 7 according to of the present invention, 19-29,29a-29u, the functional equivalent that nucleotide sequence obtained shown in 363-385 was capable be listed as according to of the present invention as Table II A or IIB the 5th or 7,19-29,29a-29u, one of polypeptide had at least 30% shown in 363-385 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II A or IIB the 5th or 7 be listed as, 19-29,29a-29u, the polypeptide of the essentially identical character of polypeptide shown in 363-385 is capable.

[0178.0.0.3] sees [0178.0.0.0]

[0179.0.3.3] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in), and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, capable, the preferred Table II B the 7th of 363-385 be listed as, 19-29,29a-29u, 363-385 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through to introduce sudden change in the encoding sequence of the sequence of standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) in Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.

[0180.0.0.3] to [0183.0.0.3]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.3.3] is employed to have as Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 is capable, preferred Table I B the 7th row, 19-29,29a-29u, the nucleotide sequence homologous compound of sequence shown in 363-385 is capable, perhaps come Table I A or IB the 5th or 7 row freely, 19-29,29a-29u, 363-385 is capable, preferred Table I B the 7th row, 19-29,29a-29u, the homologous compound of the nucleotide sequence of sequence shown in 363-385 is capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as sequence shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.3.3] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises as any sequence shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable.In one embodiment, the preferred nucleic acid molecule comprises other Nucleotide that does not show in the least possible, the arbitrary sequence shown in capable as Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method is with identical as the sequence shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable.

The employed nucleic acid molecule encoding of [0186.0.3.3] also preferred the inventive method comprises as Table II A or IIB, polypeptide of sequence shown in the 5th or 7 row, capable, preferred Table II B the 7th row of 19-29,29a-29u, 363-385,19-29,29a-29u, 363-385 are capable.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the coded polynucleotide that are used for the inventive method are with identical as sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table II B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable.

[0187.0.3.3] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as polypeptide of sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table II B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable and contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with identical as the encoding sequence of sequence shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable.

The polypeptide (=protein) that [0188.0.3.3] still has the polypeptide basic enzyme activity of the present invention (being its active basic reduction that do not have) of giving fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, actively do not reduce substantially with comparing as polypeptide expressed activity shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable and under the same conditions.

[0189.0.3.3] be the homologous compound in capable as Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.3] to [0203.0.0.3]: see that [0190.0.0.0] is to [0203.0.0.0]

[0204.0.0.3] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as polypeptide or its segmental nucleic acid molecule shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table II B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable, described nucleic acid molecule is given fine chemicals quantity in biological or its part increase;

(b) comprise, preferably comprise mature form at least as nucleic acid molecule or its segmental nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable, described nucleic acid molecule is given the increase of fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 7th row, 19-29,29a-29u, 363-385 primer or primer the nucleic acid molecule that amplification from cDNA library or genomic library is obtained in capable, and give the increase of fine chemicals quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 19-29,29a-29u, 363-385 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described peptide coding is as the structural domain of the polypeptide shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or Table I A or IB the 5th or 7 row, 19-29,29a-29u, nucleic acid molecule shown in 363-385 is capable or coding (optimized encoding is mature form at least) Table II A or IIB the 5th or 7 row, 19-29,29a-29u, at least the 15nt of the nucleic acid molecule of polypeptide shown in 363-385 is capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A the 5th or 7 row, 19-29,29a-29u, 363-385 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule not by Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in and the sequence described form.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 19-29,29a-29u, the 363-385 sequence shown in capable is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In other embodiments, nucleic acid molecule is not encoded as the sequence of polypeptide shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.Therefore, in one embodiment, the polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable at least one or the different polypeptide of a plurality of amino acid.In another embodiment, Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in nucleic acid molecule coding schedule IIA or IIB the 5th or 7 row, 19-29,29a-29u, the 363-385 protein sequence shown in capable not.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable at least 30% identical and with Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.3] to [0226.0.0.3]: see that [0205.0.0.0] is to [0226.0.0.0]

[0227.0.3.3] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I A or IB the 5th or 7 row, 19-29,29a-29u, the capable sequence or derivatives thereof of mentioning of 363-385, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of L-Methionin, L-Threonine and/or L-methionine(Met) or L-leucine and/or Isoleucine and/or Xie Ansuan biosynthetic pathway of at least one amino acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.3] to [0230.0.0.3]: see that [0228.0.0.0] is to [0230.0.0.0]

[0231.0.0.3] is in another advantageous embodiment of the inventive method, the employed biology of present method is leucine and/or Isoleucine and/or the Xie Ansuan protein that has weakened degraded simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.3] to [0282.0.0.3]: see that [0232.0.0.0] is to [0282.0.0.0]

[0283.0.3.3] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the proteinic antibody of b4054 and/or b4327 or anti-as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, the antibody of polypeptide shown in 363-385 is capable, it can utilize polypeptide of the present invention or its fragment (being polypeptide of the present invention) to produce by standard technique.Monoclonal antibody preferably.

[0284.0.0.3]: see [0284.0.0.0]

[0285.0.3.3] the present invention relates to have as sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable in one embodiment.

[0286.0.3.3] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV, 19-29,29a-29u, consensus sequence shown in 363-385 is capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV, 19-29,29a-29u, consensus sequence shown in 363-385 is capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can be by arbitrary aminoacid replacement, perhaps in other embodiments, replaced and/or lack.

[0287.0.0.3] to [0290.0.0.3]: see that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.3.3] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, sequence shown in 363-385 is capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, sequence shown in 363-385 is capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.

[0292.0.0.3]: see [0292.0.0.0]

[0293.0.3.3] in one embodiment, the present invention relates in biological or its part, give the polypeptide that fine chemicals increases, described polypeptide by nucleic acid molecule of the present invention or the employed nucleic acid molecule encoding of the present invention of the inventive method and have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help to form as the sequence of nucleic acid molecule encoding shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.

[0294.0.3.3] in one embodiment, the present invention relates to have YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the polypeptide of b4054 and/or b4327 protein active, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, sequence shown in 363-385 is capable.

[0295.0.0.3] to [0297.0.0.3]: see that [0295.0.0.0] is to [0297.0.0.0]

The statement that [0297.0.3.3] " do not contain cellular material " substantially comprises the wherein preparation of protein isolating polypeptide of the present invention from the cellular component of natural or its cell of reorganization generation.In one embodiment, the statement that " does not contain cellular material substantially " comprises and contains the preparation that is lower than about 30% (dry weight) " contaminating protein matter ", " the contaminating protein matter " more preferably less than about 20%, also more preferably less than about 10% " contaminating protein matter ", and most preferably be lower than the preparation of about 5% " contaminating protein matter ".Term " contaminating protein matter " relates to the polypeptide that is not polypeptide of the present invention.When reorganization produces polypeptide of the present invention or its biologically-active moiety, also preferably do not contain substratum substantially, promptly substratum less than the protein formulation cumulative volume 20%, be more preferably less than 10%, and most preferably less than 5%.The statement that " does not contain precursor or other chemical substantially " comprises polypeptide wherein of the present invention isolating preparation from the precursor that participates in protein synthesis or other chemical.The statement that " does not contain precursor or other chemical substantially " comprises and contains precursor or the non-YDL127W that is lower than 30% (dry weight), YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the chemical of b4054 and/or b4327, preferably be lower than 20% precursor or non-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the chemical of b4054 and/or b4327, also more preferably less than 10% precursor or non-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the chemical of b4054 and/or b4327, and most preferably be lower than 5% precursor or non-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the preparation of the chemical of b4054 and/or b4327.In preferred embodiments, isolating protein or its biologically-active moiety do not derive from the contaminating protein matter of the same biology that produces polypeptide of the present invention.Generally produce this proteinoid by recombinant technology.

The chemical of [00297.1.0.3] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 19-29,29a-29u, 363-385 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.

[0298.0.3.3] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable.

[0299.0.3.3] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of aminoacid sequence shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as the nucleotide sequence or the coded aminoacid sequence of its homologue of Table I A or IB the 5th or 7 row, 19-29,29a-29u, nucleotide sequence hybridization (preferably hybridize under stringent condition) that 363-385 is capable.

[0300.0.3.3] therefore, and be described in detail as this paper, and polypeptide of the present invention is because natural variation or mutagenesis are can be on aminoacid sequence capable different with Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385.Therefore, this polypeptide contain with Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 capable in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.3]: see [0301.0.0.0]

The biologically-active moiety of [0302.0.3.3] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II A or IIB the 5th or 7 row, 19-29,29a-29u, the aminoacid sequence of the aminoacid sequence shown in 363-385 is capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.3]: see [0303.0.0.0]

[0304.0.3.3] operation nucleic acid molecule of the present invention may cause producing and wild-type YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or the different YDL127W of b4327 protein, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.3]: see [0305.0.0.0]

[0305.0.3.3] causes that polypeptide of the present invention or polypeptide that the inventive method is used improve described active any mutagenesis strategy and all be meant nonrestrictively, and the variation of these strategies is that those skilled in the art are conspicuous.Use these the strategy and in conjunction with mechanism disclosed herein, can utilize nucleic acid molecule of the present invention and polypeptide to produce plant or its part, described plant or its part are expressed encoding wild type YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the YDL127W of b4054 and/or b4327 protein or sudden change, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, proteinic nucleic acid molecule of the present invention of b4054 and/or b4327 and peptide molecule are to improve the yield of required compound, output and/or generation efficient.

[0306.0.0.3] to [0308.0.0.3]: see that [0306.0.0.0] is to [0308.0.0.0]

[0309.0.3.3] in one embodiment, " YDL127W; YDR245W; YDR271C; YER173W; YGR101W; YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein (=polypeptide) " be meant the polypeptide that has corresponding polypeptide of the present invention or be used for the amino acid sequence of polypeptide of the inventive method; and " non-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 polypeptide " or " other polypeptide " be meant the polypeptide of aminoacid sequence with corresponding following proteins; described protein and polypeptide of the present invention be homology not basically; preferably with have YDL127W; YDR245W; YDR271C; YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the polypeptide of b4054 and/or b4327 protein active is homology not basically, does not for example give described in the literary composition active and from the protein of identical or different biology.

[0310.0.0.3] to [0334.0.0.3]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.3.3] confirmed that the dsRNAi method to the expression that reduces the capable nucleotide sequence of Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 nucleotide sequence and/or the expression of its homologue double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change in capable.In the double stranded rna molecule of the coded protein expression of the nucleotide sequence that is used for reducing one of Table I A or IB the 5th or 7 row, 19-29,29a-29u, the capable sequence of 363-385 or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.3] to [0342.0.0.3]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.3.3] is as describing, in order to cause effective reduction of expression, between the genetic transcription thing of the capable nucleotide sequence of dsRNA and Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example the dsRNA that begins to produce as the capable sequence of Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 or its homologue in a kind of organism can be used for suppressing expressing accordingly in another organism.

[0344.0.0.3] to [0361.0.0.3]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.3.3] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or its part fine chemicals increase) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, (for example coding has YDL127W for carrier of the present invention or code book invention polypeptide, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the polypeptide of b4054 and/or b4327 protein active) nucleic acid molecule.Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, cell activity improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Has YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the transgenosis of the polypeptide of b4054 and/or b4327 protein active is meant in the text owing to genomic regulation and control or manipulation, YDL127W in cell or biological or its part, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 activity or YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327-sample are active to be improved.Example and the inventive method are described in above.

[0363.0.0.3]: see [0363.0.0.0]

[0364.0.3.3] is when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---for example naturally occurring YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 bak promoter and corresponding YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, the combination of b4054 and/or b4327 protein gene---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.3] to [0382.0.0.3]: see that [0365.0.0.0] is to [0382.0.0.0]

[0383.0.3.3] is in order to prepare the fine chemicals that contains the branched-amino acid compound; fine chemicals particularly, for example isovaleric acid, isopropylmolic acid, oxo isocaproic acid, isovaleryl compound, methylvaleric acid, isopropylformic acid, methylbutyryl compound, isopropyl acid, different propionyl compound or other organic branched-chain amino acid precursor compound are originated as branched-chain amino acid can to use the compound that organically contains branched-chain amino acid.

[0384.0.0.3] to [0392.0.0.3]: see that [0384.0.0.0] is to [0392.0.0.0]

[0393.0.3.3] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(g) sample that will comprise candidate gene (be coded in and give the gene product that fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(h) identify the nucleic acid molecule of particularly under lax stringent condition, hybridizing, and randomly separate full length cDNA clone or complete genome group clone as the nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 19-29,29a-29u, capable, preferred Table I B the 7th row of 363-385,19-29,29a-29u, 363-385 are capable with nucleic acid molecule of the present invention;

(i) the candidate nucleic acid molecule is introduced the host cell that is fit to produce fine chemicals, preferred plant cell or microorganism;

(j) in host cell, express the nucleic acid molecule of being identified;

(k) level of fine chemicals in the analysis host cell; With

(l) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.3] to [0398.0.0.3]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.3.3] in one embodiment, the present invention relates to identify the method for giving the compound that the fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(c) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(d) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under the fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving fine chemicals generation raising under these conditions.

It is contemplated that the fine chemicals that screens raising produces by for example seeking the resistance of blocking-up Isoleucine synthetic medicine and observing this effect and have the active biophase much at one of high or low YDL127W than whether being that YDL127W is dependent.

[0400.0.0.3] to [0430.0.0.3]: see that [0400.0.0.1] is to [0430.0.0.0]

[0431.0.3.3] embodiment 1: clone SEQ ID No:899 in intestinal bacteria

[0432.0.3.3] uses the method for known good foundation (to consult as Sambrook, J. etc. (1989) " Molecular Cloning:A Laboratory Manual " Cold Spring HarborLaboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Mole-cular Biology ", John Wiley ﹠amp; Sons), with SEQ ID NO:899 be cloned into plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl Acad.Sci.USA, 75:3737-3741); PACYC177 (Change﹠amp; Cohen (1978) J.Bacteriol.134:1141-1156); PBS series plasmid (pBSSK+, pBSSK-and other; Stratagene, LaJolla, USA) or clay such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J.Rosenthal, A., and Waterson, R.H. (1987) Gene 53:283-286) with at expression in escherichia coli.

[0433.0.0.3] to [0460.0.0.3]: see that [0433.0.0.0] is to [0460.0.0.0]

[0461.0.3.3] embodiment 10: clone SEQ ID NO:899 is used for expressing plant

[0462.0.0.3]: see [0462.0.0.0]

[0463.0.3.3] passes through pcr amplification SEQ ID NO:899 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.3] to [0466.0.0.3]: see that [0464.0.0.0] is to [0466.0.0.0]

[0467.0.3.3] selects following primer sequence for genes of SEQ ID NO:899:

I) forward primer (SEQ ID No:911)

atgtcaaact?acgaagcctt?gctg

Ii) reverse primer (SEQ ID No:912)

tcacagggcg?cgctttacta?aaat

[0468.0.0.3] to [0479.0.0.3]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.3.3] embodiment 11: express the generation of the transgenic plant of SEQ ID No:899

[0481.0.0.3] to [0513.0.0.3]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.3.3] selects as another kind of, as people such as Geigenberger (Plant Cell﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect amino acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??YDL127W	Isoleucine	??GC	??1.33	??1.33
??YDR245W	Leucine	??GC	??1.5	??2.73
					??YDR271C	Isoleucine	??GC	??1.3	??1.74
??YER173W	Xie Ansuan	??GC	??1.18	??1.82
					??YER173W	Leucine	??GC	??1.93	??2.87
??YGR101W	Xie Ansuan	??GC	??1.47	??1.6
					??YJL072C	Isoleucine	??GC	??1.31	??4.56
??YKR057W	Xie Ansuan	??GC	??1.19	??2
					??YNL135C	Leucine	??GC	??1.45	??3.8
??YFL013C	Xie Ansuan	??GC	??1.22	??1.58

??b0149	Leucine	??GC	??1.62	??1.99
					??b0161	Xie Ansuan	??GC	??1.29	??4.99
??b0161	Isoleucine	??GC	??1.98	??11.3
					??b0161	Leucine	??GC	??1.71	??12.4
??b0486	Xie Ansuan	??GC	??1.22	??2.81
					??b1313	Xie Ansuan	??GC	??1.22	??1.5
??b1343	Xie Ansuan	??GC	??1.21	??1.23
					??b1463	Isoleucine	??GC	??1.39	??2.19
??b1463	Leucine	??GC	??1.66	??2.57
					??b2022	Xie Ansuan	??GC	??1.22	??1.26
??b2414	Xie Ansuan	??GC	??1.35	??2.39
					??b2664	Isoleucine	??GC	??1.42	??12.7
??b2664	Leucine	??GC	??1.67	??11.5
					??b3117	Isoleucine	??GC	??1.47	??7.56
??b3256	Xie Ansuan	??GC	??1.27	??1.36
					??b3938	Xie Ansuan	??GC	??1.24	??1.42
??b3983	Xie Ansuan	??GC	??1.24	??3.02
					??b4054	Leucine	??GC	??2.26	??2.63
??b4327	Isoleucine	??GC	??1.42	??1.54

[0515.0.0.3]: to [0552.0.0.3]: see that [0515.0.0.0] is to [0552.0.0.0]

[0552.1.0.3]: embodiment 15: as described in embodiment 14c, transform the Zea mays plant from Zea mays metabolite profile information.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF_ title	Metabolite	??Min	??Max
				??YKR057W	Xie Ansuan	??1.29	??1.62
??YIL150C	Xie Ansuan	??1.89	??3.26
				??YIL150C	Isoleucine	??1.79	??2.98

Table 2 shows that Xie Ansuan and/or Isoleucine have increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YKR057W or YIL150C.

Under the situation that the activity of yeast saccharomyces cerevisiae protein YKR057W or the biological ribosomal protein that takes place and translate, be similar to the S21 ribosomal protein of participation rrna or its homologue is enhanced in maize plant, preferably, giving the fine chemicals Xie Ansuan is increased between 29% and 62%.

In one embodiment, under the situation that yeast saccharomyces cerevisiae protein YIL150C or its homologue are enhanced as the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue in maize plant, preferably, give the fine chemicals Xie Ansuan be increased in 89% and 226% between and the fine chemicals Isoleucine be increased between 79% and 198%.

[00552.2.0.3]: see [00552.2.0.0]

[00553.0.3.3]

1. produce the method for leucine and/or Isoleucine and/or Xie Ansuan, it comprises:

(a) raising or generation YDL127W in non-human being or its one or more parts, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 activity of proteins; With

(b) in allowing described biology, cultivate this biology under the condition of generation leucine and/or Isoleucine and/or Xie Ansuan.

2. produce the method for leucine and/or Isoleucine and/or Xie Ansuan, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable, and described nucleic acid molecule is given in biology or its part leucine and/or Isoleucine and/or Xie Ansuan quantity to be increased;

B) contain just like Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of leucine and/or Isoleucine and/or Xie Ansuan quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of leucine and/or Isoleucine and/or Xie Ansuan quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that leucine in biology or its part and/or Isoleucine and/or Xie Ansuan quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 19-29,29a-29u, 363-385 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives in biology or its part leucine and/or Isoleucine and/or Xie Ansuan quantity and increase;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part leucine and/or Isoleucine and/or Xie Ansuan quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 19-29,29a-29u, 363-385 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded leucine and/or Isoleucine and/or Xie Ansuan.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded leucine and/or the Isoleucine and/or the Xie Ansuan of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, 19-29,29a-29u, 363-385 are capable, and described nucleic acid molecule is given in biology or its part leucine and/or Isoleucine and/or Xie Ansuan quantity to be increased;

B) contain just like Table I A or IB the 5th or 7 row, 19-29,29a-29u, 363-385 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of leucine and/or Isoleucine and/or Xie Ansuan quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of leucine and/or Isoleucine and/or Xie Ansuan quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that leucine in biology or its part and/or Isoleucine and/or Xie Ansuan quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 19-29,29a-29u, 363-385 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part leucine and/or Isoleucine and/or Xie Ansuan quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 19-29,29a-29u, 363-385 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A the 5th or 7 row, 19-29,29a-29u, 363-385 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 19-29,29a-29u, 363-385 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in leucine and/or Isoleucine and/or Xie Ansuan quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part leucine and/or Isoleucine and/or Xie Ansuan quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps leucine and/or Isoleucine and/or Xie Ansuan level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the leucine that will measure and/or Isoleucine and/or Xie Ansuan level or expression of polypeptides level and the standard leucine of when described candidate compound or the sample that comprises described multiple compound lack, measuring and/or Isoleucine and/or Xie Ansuan or expression of polypeptides level; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that leucine in plant or the microorganism and/or Isoleucine and/or Xie Ansuan produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of leucine and/or Isoleucine and/or Xie Ansuan quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of expression of polypeptides of leucine in biology or its part and/or Isoleucine and/or the increase of Xie Ansuan quantity and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that leucine in the cell and/or Isoleucine and/or Xie Ansuan produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that leucine and/or Isoleucine and/or Xie Ansuan increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce leucine and/or Isoleucine and/or Xie Ansuan;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) leucine and/or Isoleucine and/or the Xie Ansuan level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give leucine and/or Isoleucine and/or the raising of Xie Ansuan level in the host cell after it is expressed with wild-type.

20. identify and to give the method that leucine in the cell and/or Isoleucine and/or Xie Ansuan produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that leucine in biology or its part and/or Isoleucine and/or Xie Ansuan quantity or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce leucine and/or Isoleucine and/or Xie Ansuan;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) leucine and/or Isoleucine and/or the Xie Ansuan level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give leucine and/or Isoleucine and/or the raising of Xie Ansuan level in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that leucine and/or Isoleucine and/or Xie Ansuan increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control leucine and/or Isoleucine and/or Xie Ansuan level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make anti-leucine and/or Isoleucine and/or the Xie Ansuan synthetic weedicide of suppressing of plant.

[00554.0.0.3] summary: see [00554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.4] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.4] to [0008.0.0.4]: see that [0001.0.0.0] is to [0008.0.0.0]

[0009.0.4.4] as mentioned above, indispensable amino acid is that people and many Mammalss such as domestic animal are necessary.Arginine is participant's physiology and the multiextent semi-dispensable amino acid of metabolism.Why thinking that arginine is optional, is because the people can be from glutamine, L-glutamic acid and proline(Pro) de novo synthesis arginine.Yet the diet picked-up remains the main determining factor of blood plasma arginine-level, and this is to consume or insufficient supply because arginic biosynthesizing speed can not be increased to compensation.In neonatal pig, the arginic picked-up of diet is being regulated from the arginine of the synthetic whole health of proline(Pro).Arginine synthetic maximum rate (0.68g/kg/d) is not enough to supply whole health to arginic metabolic demand in piggy.In animal, L-glutamic acid works as neurotransmitter and activates glutamate receptor cationic channel (iGluR), and this has triggered electrical signal cascade or Ca ²⁺Signal cascade.In plant, amino acid involved in plant nitrogen state and plant nitrogen: the signal transduction of carbon ratio.The endogenous glutamine participates in feedback inhibition (Rawat etc., Plant Journal 19:143-152,1999 of root N picked-up by the gene transcription that suppresses coding inorganic nitrogen transporter; Zhuo etc., Plant Journal 17:563-568,1999).The non-essential amino acid proline(Pro) is synthetic from L-ornithine or L-L-glutamic acid.Proline(Pro) from the L-ornithine is relevant with protein metabolism in ornithine cycle, and relevant with sugar metabolism from the proline(Pro) of L-L-glutamic acid.Collagen is the main proline(Pro) storage vault of body.Vitamins C can be used for the treatment of collagenosis with proline(Pro).

[0010.0.0.4] to [0011.0.0.4]: see [0010.0.0.0] to [0011.0.0.0]:

[0012.0.4.4] the objective of the invention is to develop the method for the economy of synthesizing arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro), preferred L-arginine and/or L-L-glutamic acid and/or L-glutaminate and/or L-proline(Pro).

[0013.0.0.4]: see [0013.0.0.0]

[0014.0.4.4] therefore, in first embodiment, the present invention relates to produce the method for fine chemicals, wherein fine chemicals is arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro), preferred L-arginine and/or L-L-glutamic acid and/or L-glutaminate and/or L-proline(Pro).Therefore, in the present invention, term " fine chemicals " is meant " arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro) " as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro) as used herein.

[0015.0.4.4] an embodiment, term " fine chemicals " meaning is arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro), preferred L-arginine and/or L-L-glutamic acid and/or L-glutaminate and/or L-proline(Pro).In whole specification sheets, term " fine chemicals " meaning be free form or be bonded to proteinic arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro), preferred L-arginine and/or L-L-glutamic acid and/or L-glutaminate and/or L-proline(Pro), its salt, ester or acid amides.In preferred embodiments, term " fine chemicals " meaning be free form or be bonded to proteinic arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro), preferred L-arginine and/or L-L-glutamic acid and/or L-glutaminate and/or L-proline(Pro) or its salt.

[0016.0.4.4] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, increase or produce one or more YDR316W, YHR130C, YKR057W, YNL090W, b1829, b0695, b1284, b2095, b0161, b2307 and/or b3936 activity of proteins, perhaps have by the activity of proteins of nucleic acid molecule encoded polypeptide sequence shown in Table II the 5th or 7 row, 30-37,390,405 and/or 430 row and

(b) in that to allow fine chemicals be arginine or comprise under the condition that arginic fine chemicals produces in biology and make described biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more YBR204C, YFL013C, YGR104C, YPR024W, YPR133W-A, b0730, b0050, b0057, b0161, b1343, b1693, b1736, b1738, b1896, b2307, b2710, b2818, b3074, b3116, b3169, b3619, b3791b4346 and/or YFL019C activity of proteins, perhaps have by Table II the 5th or 7 row, 38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 the row shown in nucleic acid molecule encoded polypeptide sequence activity of proteins and

(b) be to make described biological growth under the condition that in biology, produces of L-glutamic acid or the fine chemicals that comprises L-glutamic acid allowing fine chemicals.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more YBR030W, YDL106C, YDR271C, YEL045C, YER173W, YFL050C, YGR135W, YIL150C, YNL090W, YPR138C, b0730, b2699, b1827, b0138, b0149, b1360, b2553, b2664, b3644 and/or b3919 activity of proteins, perhaps have by the activity of proteins of nucleic acid molecule encoded polypeptide sequence shown in Table II the 5th or 7 row, 44-56,388,389,398,411,412,425 and/or 429 row and

(b) be to make described biological growth under the condition that in biology, produces of proline(Pro) or the fine chemicals that comprises proline(Pro) allowing fine chemicals.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more YER173W, YFR042W, YKR057W, b1829, b1852, b4265, b0161, b0486, b0849, b0970, b1343, b1886, b1926, b2414, b2426, b2489, b2553, b2818, b3064, b3160, b3166, b3169, b3231, b3680, b3719, b4004, b4074 and/or b4133 activity of proteins, perhaps have by Table II the 5th or 7 row, 57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in nucleic acid molecule encoded polypeptide sequence activity of proteins and

(b) be to make described biological growth under the condition that in biology, produces of glutamine or the fine chemicals that comprises glutamine allowing fine chemicals.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, 30-62 is capable and/or 386-4352 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 the row, 30-62 is capable and/or 386-4352 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) under the condition that allows generation fine chemicals, particularly arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro), make biological growth.

[0016.1.4.4] therefore, term " fine chemicals " refers to and Table I-IV 30-37 in one embodiment, 390, relevant " arginine " or its homologue of the listed full sequence of 405 and/or 430 row, and refer in one embodiment and Table I-IV 38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427, relevant " L-glutamic acid " or its homologue of the listed full sequence of 434 and/or 435 row, and refer in one embodiment and Table I-IV 44-56,388,389,398,411,412, relevant " proline(Pro) " or its homologue of the listed full sequence of 425 and/or 429 row, and refer in one embodiment and Table I-IV 57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or relevant " glutamine " or its homologue of the capable listed full sequence of 431-433.

Therefore, in one embodiment, term " fine chemicals " refers to and Table I-IV the 43rd and relevant " L-glutamic acid " and " proline(Pro) " of the 54 listed full sequences of row, in one embodiment, term " fine chemicals " refers to and Table I-IV the 32nd and 59 row and the 34th and 60 row and relevant " arginine " and " glutamine " of the 390th and 392 listed full sequences of row, in one embodiment, term " fine chemicals " refers to and Table I-IV the 57th and 48 row and relevant " glutamine " and " proline(Pro) " of the 410th and 411 listed full sequences of row, in one embodiment, term " fine chemicals " refers to and Table I-IV the 390th and 391 row and relevant " arginine " and " L-glutamic acid " of the 405th and 406 listed full sequences of row, in one embodiment, term " fine chemicals " refers to and Table I-IV the 391st and 392 row and the 396th and 397 row and the 414th and 415 row and the 421st and 422 row and relevant " L-glutamic acid " and " glutamine " of the 427th and 428 listed full sequences of row, in one embodiment, term " fine chemicals " refers to and Table I-IV the 390th and relevant " arginine " and " L-glutamic acid " of the 391 and 392 listed full sequences of row.

Therefore, term " fine chemicals " can refer to " arginine " and/or " L-glutamic acid " and/or " glutamine " and/or " proline(Pro) " according to environment and context.For the meaning of illustrating term " fine chemicals " is meant " arginine " and/or " L-glutamic acid " and/or " glutamine " and/or " proline(Pro) ", also can use term " each fine chemicals ".

[0017.0.0.4] to [0018.0.0.4]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.4.4] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist to as Table II the 3rd be listed as, protein shown in the capable and/or 386-4352 of 30-62 is capable or by as Table I the 5th or 7 row, the coded activity of proteins of nucleic acid molecule was carried out above-mentioned modification shown in the capable and/or 386-4352 of 30-62 was capable.

[0020.0.4.4] is surprisingly found out that, at least a as Table II the 3rd row, for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine the yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana shown in capable give each fine chemicals content raising of the plant that transforms, and/or at least aly be listed as Table II the 3rd, for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or e. coli k12 protein in Arabidopis thaliana the transgene expression of 431-433 shown in capable each fine chemicals content of giving the plant that transforms improve.

Therefore, be surprisingly found out that e. coli k12 protein transgene expression in Arabidopis thaliana is given L-glutamic acid and/or the raising of proline(Pro) (or each fine chemicals) content of institute conversion plant shown in Table II the 5th row, the 43rd and 54 row.Therefore, in one embodiment, described protein or its homologue are used to produce L-glutamic acid; In one embodiment, described protein or its homologue are used to produce proline(Pro); In one embodiment, described protein or its homologue are used to produce one or more fine chemicals that are selected from L-glutamic acid and/or proline(Pro).

Therefore, be surprisingly found out that, shown in Table II the 5th row, the 43rd and 54 row and/or the 390th and 392 row e. coli k12 protein and/or shown in Table II the 5th row, the 32nd and 59 row yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana give arginine and/or the raising of glutamine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce arginine; In one embodiment, described protein or its homologue are used to produce glutamine; In one embodiment, described protein or its homologue are used to produce one or more fine chemicals that are selected from arginine and/or glutamine.

Be surprisingly found out that, shown in Table II the 5th row, the 48th and 57 row yeast saccharomyces cerevisiae protein and/or shown in Table II the 5th row, the 411st and 410 row e. coli k12 protein transgene expression in Arabidopis thaliana give proline(Pro) and/or the raising of glutamine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce proline(Pro); In one embodiment, described protein or its homologue are used to produce glutamine, and in one embodiment, described protein or its homologue are used to produce one or more fine chemicals that are selected from proline(Pro) and/or glutamine.

Be surprisingly found out that e. coli k12 protein transgene expression in Arabidopis thaliana is given L-glutamic acid and/or the raising of glutamine (or each fine chemicals) content of institute conversion plant shown in Table II the 5th row, the 391st and 392 row and/or the 396th and 397 row and/or the 414th and 415 row and/or the 421st and 422 row and/or the 427th and 428 row.Therefore, in one embodiment, described protein or its homologue are used to produce L-glutamic acid; In one embodiment, described protein or its homologue are used to produce glutamine, and in one embodiment, described protein or its homologue are used to produce one or more fine chemicals that are selected from L-glutamic acid and/or glutamine.

Be surprisingly found out that e. coli k12 protein transgene expression in Arabidopis thaliana is given institute conversion plant shown in Table II the 5th row, the 390th and 391 row and the 405th and 406 row arginine and/or L-glutamic acid (or each fine chemicals) content improve.Therefore, in one embodiment, described protein or its homologue are used to produce arginine, in one embodiment, described protein or its homologue are used to produce L-glutamic acid, and in one embodiment, described protein or its homologue are used to produce arginine and L-glutamic acid.

Be surprisingly found out that e. coli k12 protein transgene expression in Arabidopis thaliana is given arginine and/or L-glutamic acid and/or the raising of glutamine (or each fine chemicals) content of institute conversion plant shown in Table II the 5th row, the 390th and 391 and 392 row.Therefore, in one embodiment, described protein or its homologue are used to produce arginine, and in one embodiment, described protein or its homologue are used to produce L-glutamic acid; In one embodiment, described protein or its homologue are used to produce glutamine, and in one embodiment, described protein or its homologue are used to produce one or more fine chemicals that are selected from arginine and/or L-glutamic acid and/or glutamine.

[0021.0.0.4]: see [0021.0.0.0]

The sequence of [0022.0.4.4] e. coli k12 b0695 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be sensibility histidine kinase in the two-pack regulator control system.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of sensibility histidine kinase homology superfamily, preferably having a purposes of the active protein of sensibility histidine kinase in the two-pack regulator control system or its homologue, as described its to be used for producing fine chemicals in biological or its part be arginine, particularly increase arginine, the preferred arginic quantity of free or combining form.

The sequence of e. coli k12 b0730 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be that succinic thiokinase operon transcriptional and acyl are replied regulatory gene.Therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of transcriptional GntR superfamily from colibacillary, preferably has the purposes that succinic thiokinase operon transcriptional or lipid acid are replied the active protein of regulatory gene or its homologue, it is used for to be L-glutamic acid and/or proline(Pro), particularly to increase L-glutamic acid and/or proline(Pro) at biological or its part generation fine chemicals as described, L-glutamic acid particularly, proline(Pro) particularly, particularly L-glutamic acid and proline(Pro), preferred free or the L-glutamic acid of combining form and/or the quantity of proline(Pro).

The sequence of e. coli k12 b1284 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be supposition have the transcriptional (DeoR family) of DNA in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of the albumen gutR superfamily of adjusting, having tool DNA in conjunction with transcriptional (DeoR family) the active protein in wing spirane structure territory or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be arginine, particularly increase arginine, the preferred arginic quantity of free or combining form.

The sequence of e. coli k12 b1827 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is defined as having the repressor protein (IclR family) of DNA in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprise as shown here from the colibacillary DNA of having in conjunction with the repressor protein (IclR family) in wing spirane structure territory or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be leucine and/or Isoleucine and/or Xie Ansuan, particularly increase leucine and/or Isoleucine and/or Xie Ansuan, preferably free or the leucine of combining form and/or the quantity of Isoleucine and/or Xie Ansuan.In one embodiment, in the methods of the invention, have DNA and be enhanced or produce, for example be enhanced or produce in conjunction with the repressor protein (IclR family) in wing spirane structure territory or the activity of its homologue from the colibacillary DNA of having in conjunction with the activity of the repressor protein (IclR family) in wing spirane structure territory.

The sequence of e. coli k12 b1827 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be transcribing and having the repressor protein (IclR family) of DNA in conjunction with wing spirane structure territory of supposition.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of acetate operon repressor superfamily, having tool DNA in conjunction with transcription repressor protein (IclR family) the active protein in wing spirane structure territory or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be arginine, particularly increase arginine, the preferred arginic quantity of free or combining form.

The sequence of e. coli k12 b1829 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the heat shock protein with protease activity.Therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of heat shock protein(HSP) htpX superfamily from colibacillary, the purposes that preferably has " heat shock protein " active protein or its homologue with protease activity, it is used for to be glutamine and/or proline(Pro), particularly to increase arginine and/or glutamine at biological or its part generation fine chemicals as described, proline(Pro) particularly, glutamine particularly, particularly proline(Pro) and glutamine, preferred free or the proline(Pro) of combining form and/or the quantity of glutamine.In one embodiment, in the methods of the invention, the activity with heat shock protein of protease activity is enhanced or produces, and for example has the heat shock protein of protease activity or the activity of its homologue is enhanced or produces from colibacillary.

The sequence of e. coli k12 b1852 has been published in Blattner etc., and science 277 (5331), 1453.1474,1997, and its activity to be defined as be glucose .6 monophosphate dehydrogenase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of glucose one 6. phosphate dehydrogenase superfamilies, preferably having a purposes of the active protein of glucose .6. phosphate dehydrogenase or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b2095 has been published in Blattner etc., science277 (5331), 1453.1474,1997, and its activity to be defined as be tagatose one 6 one phosphokinases.Therefore, in one embodiment, the inventive method comprises as shown here have dust Xi Shi mattress and belong to the active gene product of possible tagatose 6. phosphokinase gatz superfamilies, preferably have a purposes of the active protein of tagatose .6. phosphokinase or its homologue from colibacillary, as described its to be used for producing fine chemicals in biological or its part be arginine, particularly increase arginine, the preferred arginic quantity of free or combining form.

The sequence of e. coli k12 b2699 has been published in Blattner etc., science277 (5331), 1453.1474,1997, and its activity to be defined as be DNA chain exchange and the recombinant protein activity with proteolytic enzyme and nuclease.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of recombinant protein recA superfamily, preferably having the DNA chain exchange of tool proteolytic enzyme and nuclease and a purposes of the active protein of recombinant protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of e. coli k12 b4265 has been published in BIattner etc., Science277 (5331), 1453.1474,1997, and its activity to be defined as be L. idonic acid translocator.Therefore, in one embodiment, the inventive method comprises as shown here from the active gene product of the colibacillary D-of having Serine permease superfamily, preferably have a purposes of the active protein of L-idonic acid translocator or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of yeast saccharomyces cerevisiae YBR030W has been published in Feldmann etc., EMBO J., and 13 (24), 5795-5809 (1994) and Goffeau, Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.Therefore, in one embodiment, the inventive method comprises having the active gene product of yeast saccharomyces cerevisiae putative protein matter YBR030w superfamily, preferably have a purposes of the active protein of YBR030W or its homologue from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of yeast saccharomyces cerevisiae YDL106C has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity to be defined as be the homeobox transcription factor.Therefore, in one embodiment, the inventive method comprises as shown here having undefined homeobox protein, the active gene product of homeobox homologous protein matter superfamily, preferably have the purposes of " homeobox transcription factor " active protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of yeast saccharomyces cerevisiae YFR042W has been published in Goffeau etc., and Science 274 (5287), 546-547,1996 and Murakami, Y., Nat.Genet.10 (3), 261-268,1995, and its activity to be defined as be " yeast cell survival required protein ".Therefore, in one embodiment, the inventive method comprises having the possible active gene product of membranin YFR042w superfamily of yeast saccharomyces cerevisiae, preferably have the purposes of " protein that the yeast cell survival is required " active protein or its homologue from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of yeast saccharomyces cerevisiae YGR135W has been published in Goffeau etc., and Science 274 (5287), 546-547,1996 and Tettelin etc., Nature 387 (6632 supplementary issue), 81-84 (1997), and its activity to be defined as be " proteasome composition Y13 ".Therefore, in one embodiment, the inventive method comprises having the active gene product of many catalytics endopeptidase complex body chain C9 superfamily, preferably have a purposes of the active protein of proteasome composition Y13 or its homologue from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), preferred proline(Pro) is free or combining form quantity.

The sequence of yeast saccharomyces cerevisiae YHR130C has been published in Johnston etc., Science265:2077-2082 (1994), and its cytoactive is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes of YHR130C or its homologue from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be arginine, particularly increase arginine, the preferred arginic quantity of free or combining form.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Goffeau etc., Science 274 (5287), 546-547,1996 and Churcher etc., Nature 387 (6632 supplementary issue), 84-87,1997, and its activity to be defined as be that the S phase (DNA synthetic) is initial or finish necessary chromobindins.Therefore, in one embodiment, the inventive method comprises that the S phase from yeast saccharomyces cerevisiae as shown here (DNA synthetic) is initial or finishes the purposes of necessary chromobindins or its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of yeast saccharomyces cerevisiae YPR024W has been published in Goffeau etc., Science 274 (5287), 546-547,1996 and Bussey etc., Nature 387 (6632 supplementary issue), 103-105 (1997), and its activity to be defined as be the mitochondrial protein of the ATP of CDC48/PAS1/SEC18 family enzyme.Therefore, in one embodiment, the inventive method comprises as shown here having a FtsH/SEC18/CDC48-type ATP-binding domains homology from yeast saccharomyces cerevisiae; The active gene product of cell division protein ftsH superfamily, preferably has a purposes of the active protein of the ATP of CDC48/PAS1/SEC18 family enzyme mitochondrial protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of yeast saccharomyces cerevisiae YPR133W-A has been published in Goffeau etc., and Science 274 (5287), 546-547,1996 and Bussey etc., Nature 387 (6632 supplementary issue), 103-105 (1997), and its activity to be defined as be the mitochondrial outer membrane translocase.Therefore, in one embodiment, the inventive method comprises as shown here from the mitochondrial outer membrane translocase of yeast saccharomyces cerevisiae or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of yeast saccharomyces cerevisiae YPR138C has been published in Goffeau etc., and Science 274 (5287), 546-547,1996 and Bussey etc., Nature 387 (6632 supplementary issue), 103-105 (1997), and its activity to be defined as be NH ⁴⁺Transporter.In one embodiment, the inventive method comprises the ammonium transporter that has from yeast saccharomyces cerevisiae as shown here; The active gene product of ammonium transporter nrgA superfamily, preferably has a NH ⁴⁺The purposes of the active protein of transporter or its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of yeast saccharomyces cerevisiae YBR204C has been published in Goffeau etc., and Science 274 (5287), 546-547,1996 and Feldmann etc., EMBO is (24) J.13,5795-5809 (1994), and its activity to be defined as be peroxysome lipase.Therefore, in one embodiment, the inventive method comprises as shown here from the peroxysome lipase of yeast saccharomyces cerevisiae or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of YDR271C is by Le T., Johnston M., and (in March, 1996) is committed to the EMBL/GenBank/DDBJ database, by Waterston R.; (in May, 1996) and Jia Y., (in June, 1997) is committed to the EMBL/GenBank/DDBJ database, and its cytoactive is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YDR271C from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of yeast saccharomyces cerevisiae YDR316W has been published in Goffeau etc., Science 274 (5287), 546-547,1996 and Jacq etc., Nature 387 (6632 supplementary issue), 75-78 (1997), and its activity be defined as be the supposition seven beta chain family S-adenosylmethionine dependency methyltransgerases.Therefore, in one embodiment, the inventive method comprises having the active gene product of bioC homology superfamily, preferably have the protein of seven beta chain family S-adenosylmethionine dependency methyl transferase activities of supposition or a purposes of its homologue from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be arginine, particularly increase arginine, the preferred arginic quantity of free or combining form.

The sequence of yeast saccharomyces cerevisiae YEL045C is by Dietrich etc., and Nature 387:78-81 (1997) announces, and its cytoactive is not still characterized.Therefore, in one embodiment, the inventive method comprises having the active gene product of yeast belong putative protein matter YEL045c superfamily, preferably have a purposes of the active protein of YEL045C or its homologue from yeast saccharomyces cerevisiae as shown here, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of yeast saccharomyces cerevisiae YER173w has been published in Dietrich, Nature 387 (6632 supplementary issue), 78-81,1997, and Goffeau, Science 274 (5287), 546-547,1996, and its activity is defined as " participating in dna damage and reduction division pachytene stage check position activatory check position protein ".Therefore, in one embodiment, the inventive method comprises " participating in dna damage and reduction division pachytene stage check position activatory check position protein " as shown here or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine and/or proline(Pro), particularly increase glutamine and/or proline(Pro), particularly increase glutamine, particularly proline(Pro), particularly glutamine and proline(Pro), preferably free or the glutamine of combining form and/or the quantity of proline(Pro).

The sequence of yeast saccharomyces cerevisiae YFL013C has been published in Goffeau, A., and Science 274 (5287), 546-547,1996 and Murakami, Y., Nat.Genet.10 (3), 261-268,1995, and its activity to be defined as be " INO80 chromatin reconstitution complex subunit ".Therefore, in one embodiment, the inventive method comprises as shown here having the possible active gene product of membranin YFL013c superfamily of yeast saccharomyces cerevisiae, preferably have the purposes of " INO80 chromatin reconstitution complex subunit " active protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of yeast saccharomyces cerevisiae YFL050C has been published in Murakami etc.,, Nat.Genet.10 (3), 261-268,1995, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be two-trivalent inorganic cation transporter.Therefore, in one embodiment, the inventive method comprises having magnesium and the active gene product of cobalt translocator superfamily, preferably have a purposes of the active protein of two-trivalent inorganic cation transporter or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for producing the generation proline(Pro) that fine chemicals is in biological or its part as described, particularly increases proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of yeast saccharomyces cerevisiae YGR104C has been published in Thompson etc., Cell 73:1361-1375,1993, and its activity to be defined as be " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 " ".Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of yeast rna polymerase II repressor SRB5 superfamily, preferably have the purposes of " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 " active protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of yeast saccharomyces cerevisiae YKR057W has been published in Dujon etc., Nature 369 (6479), 371-378,1994 and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be to participate in that rrna is biological to be taken place and the ribosomal protein that is similar to the S21 ribosomal protein of translation.Therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of rat ribosomal protein S21 superfamily from yeast saccharomyces cerevisiae, preferably have and participate in the biological active protein of the ribosomal protein that is similar to the S21 ribosomal protein that takes place and translate of rrna or the purposes of its homologue, it is used for to be arginine and/or glutamine, particularly to increase arginine and/or glutamine at biological or its part generation fine chemicals as described, arginine particularly, glutamine particularly, particularly arginine and glutamine, preferred free or the arginine of combining form and/or the quantity of glutamine.

The sequence of e. coli k12 b0050 (accession number NP_414592) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the conservative protein matter of potential participation protein-protein interaction.Therefore, in one embodiment, the inventive method comprises as shown here from the active gene product of the colibacillary paG of having protein superfamily, preferably have the active protein of conservative protein matter of potential participation protein-protein interaction or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b0057 (accession number NP_414599) has been published in Blattner etc., and Science 277 (5331), 1453-1474, and 1997, and its activity does not characterize yet.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b0057 of having protein active or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b0138 (accession number NP_414680) has been published in Blattner etc., and Science 277 (5331), 1453-1474, and 1997, and its activity is defined as pili sample adhesin protein.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b0138 of having protein active or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of e. coli k12 b0149 (accession number NP_414691) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be bi-functional penicillin-conjugated protein 1b: glycosyltransferase (N-end); Transpeptidase (C-end).Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having penicillin-active gene product of conjugated protein 1B superfamily, preferably having bi-functional penicillin-conjugated protein 1b: glycosyltransferase (N-end); The purposes of transpeptidase (C-end) active protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of e. coli k12 b0161 (accession number NP_414691) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is pericentral siphon serine protease (heat shock protein) that its activity is defined as. therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of Helicobacterium serine protease superfamily from colibacillary, the purposes that preferably has the active protein of pericentral siphon serine protease (heat shock protein) or its homologue, it is used for to be arginine and/or L-glutamic acid and/or glutamine, particularly to increase arginine at biological or its part generation fine chemicals as described, L-glutamic acid particularly, glutamine particularly, particularly arginine and L-glutamic acid, particularly arginine and glutamine, particularly glutamine and L-glutamic acid, particularly arginine and glutamine and L-glutamic acid, preferred free or the arginine of combining form and/or the quantity of L-glutamic acid and/or glutamine.

The sequence of e. coli k12 b0486 (accession number NP_415019) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is amino acid/amine transporter (APC family) that its activity is defined as. therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of membranin ybaT superfamily from colibacillary, the purposes that preferably has amino acid/amine transporter (APC family) active protein or its homologue, it is used for to be glutamine, particularly to increase glutamine at biological or its part generation fine chemicals as described, the quantity of the glutamine of preferred free or combining form.

The sequence of e. coli k12 b0849 (accession number NP_415370) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of glutaredoxin superfamily, preferably having a purposes of the gsh dependency ribonucleotide reductase glutaredoxin active protein of 1 redox coenzyme or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b0970 (accession number NP_415490) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be glutamate receptor.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria ybhL protein superfamily, preferably having a purposes of the active protein of glutamate receptor protein matter or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b1343 (accession number NP_415490) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the ATP RNA-dependent helicase that is stimulated by 23SrRNA.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the gene product of intestinal bacteria b1343 protein active, preferably having the protein of the ATP RNA-dependent helicase activity that is stimulated by 23SrRNA or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine and/or glutamic acid, particularly increase glutamine, particularly L-glutamic acid, particularly glutamine and L-glutamic acid, the preferred quantity of the glutamine and/or glutamic acid of free or combining form.

The sequence of e. coli k12 b1360 (accession number NP_415878) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is dna replication protein matter that its activity is defined as. therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of dna replication protein matter dnaC superfamily from colibacillary, the purposes that preferably has the active protein of dna replication protein matter or its homologue, it is used for to be proline(Pro), particularly to increase proline(Pro) at biological or its part generation fine chemicals as described, the quantity of the proline(Pro) of preferred free or combining form.

The sequence of e. coli k12 b1693 (accession number NP_416208) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is the 3-dehydroquinate dehydratase that its activity is defined as. therefore, in one embodiment, the inventive method comprises as shown here from the active gene product of the colibacillary 3-of having dehydroquinate dehydratase superfamily, the purposes that preferably has the active protein of dna replication protein matter or its homologue, it is used for to be L-glutamic acid, particularly to increase L-glutamic acid at biological or its part generation fine chemicals as described, the quantity of the L-glutamic acid of preferred free or combining form.

The sequence of e. coli k12 b1736 (accession number NP_416250) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be cellobiose/arbutin/saligenin sugar-specific protein PEP dependency phosphotransferase.Therefore, in one embodiment, the inventive method comprises as shown here having a phosphotransferase system lactose specific enzymes II from colibacillary, the active gene product of factor III superfamily, preferably have the cellobiose/arbutin/saligenin sugar-protein of specific protein PEP dependency phosphate transferase activity or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b1738 (accession number NP_416252) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be PEP dependency phosphotransferase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having phosphotransferase system enzyme II cellobiose-active gene product of atopen IIB superfamily, preferably having the protein of PEP dependency phosphate transferase activity or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b1886 (accession number NP_416400) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is methyl acceptor chemotactic protein II that its activity is defined as, aspartic acid transmitter acceptor. therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of methyl acceptor chemotactic protein superfamily from colibacillary, preferably has methyl acceptor chemotactic protein II, the protein of aspartic acid transmitter receptor active or the purposes of its homologue, it is used for to be glutamine, particularly to increase glutamine at biological or its part generation fine chemicals as described, the quantity of the glutamine of preferred free or combining form.

The sequence of e. coli k12 b1896 (accession number NP_416410) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is trehalose-6-phosphate synthase that its activity is defined as. therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary α of having-trehalose-6-phosphate synthase (forming UdP) active gene product of superfamily, the purposes that preferably has the active protein of trehalose-6-phosphate synthase or its homologue, it is used for to be L-glutamic acid, particularly to increase L-glutamic acid at biological or its part generation fine chemicals as described, the quantity of the L-glutamic acid of preferred free or combining form.

The sequence of e. coli k12 b1926 (accession number NP_416436) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be flagellin fliT.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of flagellin fliT or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b2307 (accession number NP_416810) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is Histidine and Methionin/arginine/ornithine translocator (ATP-binding cassette superfamily, film) that its activity is defined as.Therefore, in one embodiment, the inventive method comprises as shown here having an active gene product of Histidine permease protein M superfamily from colibacillary, preferably has Histidine and Methionin/arginine acid/ornithine translocator (ATP-binding cassette superfamily, film) purposes of active protein or its homologue, it is used for to be L-glutamic acid and/or arginine, particularly to increase L-glutamic acid at biological or its part generation fine chemicals as described, arginine particularly, particularly L-glutamic acid and arginine, the L-glutamic acid and/or the arginic quantity of preferred free or combining form.

The sequence of e. coli k12 b2414 (accession number NP_416909) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of threonine dehydra(ta)se superfamily, preferably having a purposes of PLP dependent enzyme cysteine synthase A and the active protein of O-acetylserine sulfhydrylase A subunit or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b2426 (accession number NP_416921) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the oxydo-reductase with NAD (P)-binding domains.Therefore, in one embodiment, the inventive method comprises as shown here having a ribitol dehydrogenase from colibacillary, the active gene product of short-chain alcohol dehydrogenase homology superfamily, preferably have the protein of oxidoreductase activity of supposition or a purposes of its homologue with NAD (P)-binding domains, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b2489 (accession number NP_416984) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be hydrogenase Fe-S subunit.Therefore, in one embodiment, the inventive method comprises as shown here from the active gene product of the colibacillary psbG of having protein superfamily, preferably have a purposes of the active protein of hydrogenase Fe-S subunit or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b2553 (accession number NP_417048) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is that therefore glutamine synthetase regulates albumen P-II. that its activity is defined as, in one embodiment, the inventive method comprises as shown here having an active gene product of the albumen P-II superfamily of adjusting from colibacillary, preferably has the purposes that glutamine synthetase is regulated the active protein of albumen P-II or its homologue, it is used for to be proline(Pro) and/or glutamine, particularly to increase proline(Pro) at biological or its part generation fine chemicals as described, glutamine particularly, particularly proline(Pro) and glutamine, preferred free or the proline(Pro) of combining form and/or the quantity of glutamine.

The sequence of e. coli k12 b2664 (accession number NP_417150) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be to have the transcription repressor (GntR family) of DNA in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of transcriptional gabP superfamily, preferably having tool DNA in conjunction with active transcription repressor (GntR family) the active protein in wing spirane structure territory or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of e. coli k12 b2710 (accession number NP_417190) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be (FIRd) bi-functional NO and O of flavine rubredoxin (flavorubredoxin) ₂Reductase enzyme.Therefore, in one embodiment, the inventive method comprises as shown here having an intestinal bacteria putative protein matter b2710 from colibacillary, the rubredoxin homology, preferably has flavine rubredoxin (FIRd) bi-functional NO and an O at the active gene product of Methanobacterium (Methanobacterium) flavoprotein A superfamily ₂The protein of reductase activity or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b2818 (accession number NP_417295) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be N-acetylglutamat synthase (amino acid N-Transacetylase).Therefore, in one embodiment, the inventive method comprises as shown here having an amino-acid acetyltransferase from colibacillary, the active gene product of acetylglutamate kinase superfamily, the purposes that preferably has the active protein of N-acetylglutamat synthase (amino acid N-Transacetylase) or its homologue, it is used for to be glutamine and/or glutamic acid, particularly to increase glutamine at biological or its part generation fine chemicals as described, L-glutamic acid particularly, particularly glutamine and L-glutamic acid, preferred free or the L-glutamic acid of combining form and/or the quantity of glutamine.

The sequence of e. coli k12 b3064 (accession number NP_417536) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the O-sialoglycoprotein endopeptidase with Actin muscle sample ATP enzymatic structure territory.Therefore, in one embodiment, the inventive method comprises as shown here from the active gene product of the colibacillary O-of having sialoglycoprotein endopeptidase superfamily, preferably have the protein of O-sialoglycoprotein endopeptidase activity in Actin muscle sample ATP enzymatic structure territory or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b3074 (accession number NP_417545) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises as shown here having a secretion molecular chaperones CsaA from colibacillary, methionyl-tRNA synthetic enzyme, form the active gene product of dimeric superfamily, preferably have a purposes of protein or its homologue of tRNA synthase activity, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b3116 (accession number NP_417586) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be anaerobic inducibility L-Threonine/L-Serine permease (HAAAP family).Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having Threonine-active gene product of Serine permease superfamily, preferably having a purposes of anaerobic inducibility L-Threonine/L-Serine permease (HAAAP family) active protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b3160 (accession number NP_417629) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the monooxygenase with luciferase sample atpase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the active gene product of the colibacillary ynbW of having protein superfamily, preferably have the protein of monooxygenase activity of tool luciferase sample atpase activity or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b3166 (accession number NP_417635) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be tRNA Pseudouridine 5S synthase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of Escherichia coli protein P35 superfamily, preferably having the protein of tRNA Pseudouridine 5S synthase activity or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b3169 (accession number NP_417638) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Transcription Termination-antitermination factor.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria transcription factor nusA superfamily, preferably having a purposes of the active protein of Transcription Termination-antitermination factor or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine and/or glutamic acid, particularly increase glutamine, particularly L-glutamic acid, particularly glutamine and L-glutamic acid, preferably free or the L-glutamic acid of combining form and/or the quantity of glutamine.

The sequence of e. coli k12 b3231 (accession number NP_417698) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L13.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria ribosomal protein L13 superfamily, preferably having a purposes of the active protein of 50S ribosomal subunit protein matter L13 or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b3619 (accession number NP_418076) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be ADP-L-glycerine-D-mannoheptose-6-epimerase in conjunction with NAD (P).Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary ADP of having glycerine mannoheptose 6-epimerase, the active gene product of UDP glucose 4-epimerase homology superfamily, preferably have the ADP-L-glycerine-D-mannoheptose-active protein of 6-epimerase in conjunction with NAD (P) or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of e. coli k12 b3644 (accession number NP_418101) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the stress-induced protein that does not characterize.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of protein HI0467 superfamily, preferably having the protein of the stress-induced protein active that does not characterize or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of e. coli k12 b3680 (accession number NP_418136) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the transcriptional (AraC/XylS family) with homeodomain sample DNA binding domains.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the gene product of intestinal bacteria b3680 protein active, preferably having transcriptional (AraC/XylS family) the active protein of tool homeodomain sample DNA binding domains or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b3791 (accession number NP_418238) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be to participate in the biosynthetic transaminase of lipopolysaccharides.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of erythromycin resistance protein superfamily, preferably having the protein that participates in the biosynthetic transaminase activity of lipopolysaccharides or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine and/or glutamic acid, particularly increase glutamine, particularly L-glutamic acid, particularly glutamine and L-glutamic acid, preferably free or the L-glutamic acid of combining form and/or the quantity of glutamine.

The sequence of e. coli k12 b3919 (accession number NP_418354) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be triose-phosphate isomerase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of triose-phosphate isomerase superfamily, preferably having a purposes of the active protein of triose-phosphate isomerase or its homologue, as described its to be used for producing fine chemicals in biological or its part be proline(Pro), particularly increase proline(Pro), the preferred quantity of the proline(Pro) of free or combining form.

The sequence of e. coli k12 b3936 (accession number NP_418371) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L32.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria ribosomal protein L31 superfamily, preferably having a purposes of the active protein of 50S ribosomal subunit protein matter L32 or its homologue, as described its to be used for producing fine chemicals in biological or its part be arginine, particularly increase arginine, the preferred arginic quantity of free or combining form.

The sequence of e. coli k12 b4004 (accession number NP_418432) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be transcription regulatory protein.Therefore, in one embodiment, the inventive method comprises as shown here having nitrogen assimilation adjusting albumen ntrC or reply the regulatory gene homology from colibacillary, the active gene product of RNA polymerase delta antigen interaction domain homology superfamily, preferably has a purposes of the active protein of transcription regulatory protein or its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b4074 (accession number NP_418498) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the biological albumen that takes place of cytochrome c-type.Therefore, in one embodiment, the inventive method comprises as shown here from the active gene product of the colibacillary nrfE of having protein superfamily, preferably have the biological protein of protein-active or a purposes of its homologue of taking place of cytochrome c-type, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b4133 (accession number NP_418557) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the transcriptional activation agent (OmpR family) that pH replys.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b4133 of having protein active, preferably have transcriptional activation agent (OmpR family) the active protein that pH replys or a purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be glutamine, particularly increase glutamine, the preferred quantity of the glutamine of free or combining form.

The sequence of e. coli k12 b4346 (accession number NP_418766) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be 5-methylcytosine specificity restriction enzyme McrBC composition.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the gene product of 5-methylcytosine specificity restriction enzyme B ultrasonic family active, preferably having a purposes of the 5-methylcytosine specificity restriction enzyme active protein of McrBC composition or its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The sequence of yeast saccharomyces cerevisiae YFL019C (accession number S48324.) has been published in Murakami etc., Nat.Genet.10:261-268 (1995), and its activity does not characterize yet.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product with YFL019C protein active of yeast saccharomyces cerevisiae or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be L-glutamic acid, particularly increase L-glutamic acid, the preferred quantity of the L-glutamic acid of free or combining form.

The homologous compound (=homologue) of [0023.0.4.4] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide homologue of any one be to have same or similar active homologue respectively, particularly active increasing gives that each fine chemicals content increases in the biology.

In one embodiment, homologue be as Table I or II the 7th row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine the sequence shown in capable.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide any one homologue from eukaryote.

In one embodiment, homologue is from fungi.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide any one homologue from Ascomycota.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide any one homologue from yeast.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide any one homologue from the yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide homologue of any one be homologue from Saccharomycetes.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide homologue of any one be to have same or similar active homologue and from Saccharomycetaceae.

In one embodiment, Table II the 3rd row, be respectively for arginic 30-33 capable and/or for the 38-42 of L-glutamic acid capable and/or the 435th row and/or for the 44-53 of proline(Pro) capable and/or for the 57-59 of glutamine capable shown in the polypeptide homologue of any one be to have same or similar active homologue and come the yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given each fine chemicals content increase in the biology.

In one embodiment, homologue is to have as Table I or II the 7th row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the homologue of sequence.

In one embodiment, Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide any one homologue from bacterium.

In one embodiment, Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide any one homologue from Proteobacteria.

In one embodiment, Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide homologue of any one be to have same or similar active homologue and from γ-distortion Gammaproteobacteria.

In one embodiment, Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide any one homologue from the enterobacteria order.

In one embodiment, Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide homologue of any one be homologue from enterobacteriaceae.

In one embodiment, Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide homologue of any one be to have same or similar active homologue and from Escherichia.

[0023.1.4.4] as Table II the 3rd row, 30-62 is capable and/or 386-4352 capable shown in the homologue of polypeptide can be by Table I the 7th row, 30-62 is capable and/or 386-4352 capable shown in the polypeptide of nucleic acid molecule encoding, perhaps can be Table II the 7th row, 30-62 is capable and/or 386-4352 capable shown in polypeptide.

[0024.0.0.4]: see [0024.0.0.0]

[0025.0.4.4] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause biology or its part, arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine increase in the preferred described biomass cells, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in activity of proteins.

In preferred embodiments, protein or polypeptide have Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in proteinic above-mentioned extra activity.

In this manual, if this kind protein or polypeptide still have Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in any one proteinic biology or the enzyme activity, perhaps with Table II the 3rd row, 30-33 is capable and/or 38-42 is capable and/or 435 and/or 44-53 is capable and/or 57-59 capable shown in any one yeast saccharomyces cerevisiae protein and/or Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427 and/or 434 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in any one e. coli k12 protein compare, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, the most preferred 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.4] to [0033.0.0.4]: see that [0025.1.0.0] is to [0033.0.0.0]

[0034.0.4.4] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, it has Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein, or by Table I the 5th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the protein of nucleic acid molecule encoding, or its homologue is (as Table I the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in homologue) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows each fine chemicals quantity that increases.

[0035.0.0.4] to [0044.0.0.4]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.4.4] is at e. coli k12 protein b0695 or its homologue (shown in Table I the 5th or 7 row, the 35th row) sensibility histidine kinase (transmitter kinases composition) in the two-pack signal transduction system for example, by modifying phosphorylation, dephosphorylation modification, unspecified signal transduction, respiratory regulation, under the situation that the activity of aerobic respiration is enhanced, preferably, give in one embodiment fine chemicals, preferred arginine be increased in 51% and 319% or higher between.

Under the situation that for example be used for C-compound and carbohydrate utilization at e. coli k12 protein b0730 or its homologue (shown in Table I the 5th or 7 row, the 43rd or 54 row), transcribe control, protokaryon Nucleotide, transcription repressor, DNA is enhanced in conjunction with the activity of the transcriptional of adjusting, preferably, give in one embodiment fine chemicals be increased in 35% and 272% between, preferred L-glutamic acid be increased in 55% and 115% between and/or proline(Pro) be increased in 35% and 272% or higher between.

Under the situation that for example be used for C-compound and carbohydrate utilization at e. coli k12 protein b1284 or its homologue (shown in Table I the 5th or 7 row, the 36th row), transcribe control, transcription repressor, DNA is enhanced in conjunction with the activity of the transcriptional of regulating, preferably, give in one embodiment fine chemicals, preferred arginine be increased in 47% and 183% or higher between.

For example transcribe under the situation that the activity of the transcription repressor of control is enhanced at e. coli k12 protein b1827 or its homologue (shown in Table I the 5th or 7 row, the 56th row), preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 42% and 126% between or higher between.

(be listed as at e. coli k12 protein b1829 or its homologue as Table I the 5th or 7, the 34th or 60 the row shown in) activity be enhanced, for example participate in coercing reaction, the pheromone reaction, the mating type decision, sex-specific protein, protein modification, under the situation that the activity of the heat shock protein with protease activity (htpx) of proteolytic degradation is enhanced, preferably, giving fine chemicals in one embodiment is increased between 45% and 1141%, between preferred glutamine is increased in 50% and 68% and/or arginine be increased in 45% and 1141% or higher between.

Activity at e. coli k12 protein b1852 or its homologue (shown in Table I the 5th or 7 row, the 61st row) is enhanced, for example participate under the situation that the activity of the glucose-6-phosphate dehydrogenase (G6PD) of phosphopentose pathway oxidation branch, C-compound and carbohydrate utilization, NAD/NADP combination, nucleotide metabolism, VITAMIN, cofactor and prothetic group, energy metabolism is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 40% and 42% or higher between.

Activity at e. coli k12 protein b2095 or its homologue (shown in Table I the 5th or 7 row, the 37th row) is enhanced, for example under the situation that the activity of tagatose-6-phosphokinase is enhanced, preferably, give in one embodiment fine chemicals, preferred arginine be increased in 55% and 59% or higher between.

Activity at e. coli k12 protein b2699 or its homologue (shown in Table I the 5th or 7 row, the 55th row) is enhanced, for example participate under the situation that the activity of DNA reorganization and DNA reparation, pheromone reaction, mating type decision, sex-specific protein, Nucleotide bonded recombinant protein recA is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 32% and 141% or higher between.

Activity at e. coli k12 protein b4265 or its homologue (shown in Table I the 5th or 7 row, the 62nd row) is enhanced, for example participate under the situation that the activity of the D-Serine permease of the transhipment of C-compound and carbohydrate, C-compound and carbohydrate utilization is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 32% and 47% or higher between.

At yeast saccharomyces cerevisiae protein YBR030W or its homologue (shown in Table I the 5th or 7 row, the 44th row), for example participate in C-compound and carbohydrate utilization, phosphopentose pathway and/or transcribe under the situation that the activity of " the not profiling protein matter YBR030W " of control is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 51% and 282% or higher between.

At yeast saccharomyces cerevisiae protein YDL106C or its homologue (shown in Table I the 5th or 7 row, the 45th row), under the situation that for example participate in nucleotide metabolism adjusting, phosphoric acid utilization adjusting, transcribe control, nuclear homeobox activity of proteins is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 51% and 99% or higher between.

At yeast saccharomyces cerevisiae protein YFR042W or its homologue (shown in Table I the 5th or 7 row, the 58th row), for example under the situation that the activity of " protein that the yeast cell survival is required " is enhanced, preferably, in one embodiment, give fine chemicals, preferred glutamine be increased in 41% and 43% or higher between.

At yeast saccharomyces cerevisiae protein YGR135W or its homologue (shown in Table I the 5th or 7 row, the 50th row), for example participate under the situation that the activity of the proteasome composition Y13 of tenuigenin and nucleus degraded, endoplasmic reticulum, nucleus, cytodifferentiation, proteasome degraded is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 32% and 289% or higher between.

At yeast saccharomyces cerevisiae protein YHR130C or its homologue (shown in Table I the 5th or 7 row, the 31st row), for example under the situation that the activity of " putative protein matter YBR030W " is enhanced, preferably, give in one embodiment fine chemicals, preferred arginine be increased in 67% and 85% or higher between.

At yeast saccharomyces cerevisiae protein YIL150C or its homologue (shown in Table I the 5th or 7 row, the 51st row), for example participate in DNA synthetic and duplicate, S phase (DNA is synthetic) of mitotic cell cycle and cell cycle control is initial or finish under the situation that the activity of necessary chromobindins is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 33% and 304% or higher between.

At yeast saccharomyces cerevisiae protein YPR024W or its homologue (shown in Table I the 5th or 7 row, the 41st row), for example under the situation about being enhanced for the activity of the required ATP of the CDC48/PAS1/SEC18 family enzyme mitochondrial protein of protein complex assembling, other proteolytic degradation, plastosome, protein folding and stability, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 26% and 43% or higher between.

At yeast saccharomyces cerevisiae protein YPR133W-A or its homologue (shown in Table I the 5th or 7 row, the 42nd row), for example under the situation that the activity of the translocase of mitochondrial outer membrane is enhanced, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 34% and 68% or higher between.

At yeast saccharomyces cerevisiae protein YPR138C or its homologue (shown in Table I the 5th or 7 row, the 53rd row), for example participate in anion transport (Cl ^-, SO ₄ ^2-, PO ₄ ^3-Deng), other cation transfer (Na ⁺, K ⁺, Ca ²⁺, NH ₄ ⁺Deng), under the activity of the ammonium transporter of the transhipment of nitrogen and sulphur, cell input, the cross-cell membrane transhipment situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 54% and 520% or higher between.

At yeast saccharomyces cerevisiae protein YBR204C or its homologue (shown in Table I the 5th or 7 row, the 38th row), for example participate under the situation that the activity of the peroxysome lipase that lipid, lipid acid and isoprenoid, peroxysome decompose is enhanced, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 55% and 76% or higher between.

At yeast saccharomyces cerevisiae protein YDR271C or its homologue (shown in Table I the 5th or 7 row, the 46th row), for example under the situation that the activity of " putative protein matter YDR271C " is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 36% and 482% or higher between.

At yeast saccharomyces cerevisiae protein YDR316W or its homologue (shown in Table I the 5th or 7 row, the 30th row), for example under the situation that the activity of S-adenosylmethionine dependency methyltransgerase is enhanced, preferably, give in one embodiment fine chemicals, preferred arginine be increased in 45% and 102% or higher between.

At yeast saccharomyces cerevisiae protein YEL045C or its homologue (shown in Table I the 5th or 7 row, the 47th), for example under the situation that the activity of " putative protein matter YBR030W " is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 41% and 89% or higher between.

At yeast saccharomyces cerevisiae protein YER173w or its homologue (shown in Table I the 5th or 7 row, the 48th or 57 row), for example participate in dna damage and reduction division pachytene stage check position activatory check position protein; DNA reorganization and DNA repair, under cell cycle check position (form generation, dna damage, dna replication dna, mitotic division mutually with the check position of spindle body), nucleic acid combination, DNA synthesizes and the activity of duplicating is enhanced the situation, preferably, give in one embodiment fine chemicals be increased in 34% and 285% between, preferred glutamine be increased in 86% and 285% between and/or proline(Pro) be increased in 34% and 191% or higher between.

At yeast saccharomyces cerevisiae protein YFL013C or its homologue (shown in Table I the 5th or 7 row, the 39th row), for example under the situation that the activity of " INO80 chromatin reconstitution complex subunit " is enhanced, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 81% and 134% or higher between.

At yeast saccharomyces cerevisiae protein YFL050C or its homologue (shown in Table I the 5th or 7 row, the 49th row), for example participate under the situation that the activity of two-trivalent inorganic cation transporter of heavy metal ion transhipment (Cu, Fe etc.), cell input, detoxifcation, metal ion (Na, K, Ca etc.) homeostasis, cross-cell membrane transhipment is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 44% and 74% or higher between.

At yeast saccharomyces cerevisiae protein YGR104C or its homologue (shown in Table I the 5th or 7 row, the 40th row), for example participate under the situation that the activity of " the yeast rna polymerase II repressor SRB5 and/or the RNA polymerase B repressor SRB5 " of transcriptional activity is enhanced, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 64% and 96% or higher between.

At yeast saccharomyces cerevisiae protein YKR057W or its homologue (shown in Table I the 5th or 7 row, the 32nd or 59 row), for example participate under the situation that the activity of the biological generation of rrna, cytodifferentiation and ribosomal protein translation and similar to S21A, S26A and/or YS25 ribosomal protein is enhanced, preferably, give in one embodiment fine chemicals be increased in 41% and 457% between, preferred glutamine be increased in 41% and 284% between and/or arginine be increased in 57% and 457% or higher between.

At e. coli k12 protein b0050 or its homologue, under the activity situation about being enhanced of the conservative protein matter of for example potential participation protein-protein interaction (shown in Table II the 5th or 7 row, the 386th row), preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 37% and 97% or higher between.

At e. coli k12 protein b0057 or its homologue, for example have under the situation that the activity as institute's active protein of definition in [0022.0.4.4] (routine Table II the 5th or 7 is listed as, the 387th capable shown in) is enhanced, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 35% and 83% or higher between.

At e. coli k12 protein b0138 or its homologue, for example under the activity situation about being enhanced of pili sample adhesin protein (shown in Table II the 5th or 7 row, the 388th row), preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 50% and 180% or higher between.

At e. coli k12 protein b0149 or its homologue, bi-functional penicillin-conjugated protein 1b for example: glycosyltransferase (N-end); Under the activity situation about being enhanced of transpeptidase (C-end) (shown in Table II the 5th or 7 row, the 389th row), preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 33% and 120% or higher between.

At e. coli k12 protein b0161 or its homologue, for example the pericentral siphon serine protease is (as Table II the 5th or 7 row, shown in 390-392 is capable) activity situation about being enhanced under, preferably, give fine chemicals in one embodiment, preferred arginine be increased in 628% and 881% or more between, preferred L-glutamic acid be increased in 35% and 65% or more between, preferred glutamine be increased in 43% and 256% or more between, preferred arginine and L-glutamic acid be increased in 35% and 881% or more between, preferred arginine and glutamine be increased in 43% and 881% or more between, preferred L-glutamic acid and glutamine be increased in 35% and 256% or more between, preferred arginine and L-glutamic acid and glutamine be increased in 35% and 881% or higher between.

At e. coli k12 protein b0486 or its homologue, for example under the activity situation about being enhanced of amino acid/amine transporter (APC family) (for example shown in Table II the 5th or 7 row, the 393rd row), preferably, give in one embodiment each fine chemicals, preferred glutamine be increased in 51% and 128% or more between.

At e. coli k12 protein b0849 or its homologue, the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase for example, under the activity situation about being enhanced of (for example shown in Table II the 5th or 7 row, the 394th row), preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 37% and 50% or higher between.

At e. coli k12 protein b0970 or its homologue, for example under the activity situation about being enhanced of glutamate receptor (for example shown in Table II the 5th or 7 row, the 395th row), preferably, give in one embodiment each fine chemicals, preferred glutamine be increased in 59% and 380% or more between.

In one embodiment, (be listed as at e. coli k12 protein b1343 or its homologue as Table II the 5th or 7, the 396th and 397 the row shown in) activity be enhanced, the activity of proteins that for example participates in rRNA processing and/or translation is enhanced, preferably under the situation that the activity of the ATP RNA-dependent helicase that is stimulated by 23SrRNA or its homologue is enhanced, preferably, give each fine chemicals, preferred glutamine be increased in 37% and 39% or more between, preferably give L-glutamic acid be increased in 48% and 99% or more between, preferably give glutamine and L-glutamic acid be increased in 37% and 99% or higher between.

At e. coli k12 protein b1360 or have the active protein of dna replication protein matter or its homologue of the supposition of being defined as, for example under the activity situation about being enhanced of transcriptional (for example shown in Table II the 5th or 7 row, the 398th row), preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 33% and 70% or higher between.

At e. coli k12 protein b1693 or its homologue, for example under the activity situation about being enhanced of 3-dehydroquinate dehydratase (for example shown in Table II the 5th or 7 row, the 399th row), preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 39% and 149% or higher between.

At e. coli k12 protein b1736 or its homologue, for example under the activity situation about being enhanced of PEP dependency phosphotransferase (for example shown in Table II the 5th or 7 row, the 400th row), preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 46% and 97% or higher between.

In one embodiment, at e. coli k12 protein b1738 or have under the situation that the activity of the protein that is defined as PEP dependency phosphate transferase activity or its homologue (shown in Table II the 5th or 7 row, the 401st row) is enhanced, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 38% and 107% or higher between.

In one embodiment, at e. coli k12 protein b1886 or methyl acceptor chemotactic protein II, under the activity situation about being enhanced of aspartic acid transmitter acceptor or its homologue (shown in Table II the 5th or 7 row, the 402nd row), preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 36% and 124% or higher between.

In one embodiment, under the situation that the activity of e. coli k12 protein b1896 or trehalose-6-phosphate synthase or its homologue (shown in Table II the 5th or 7 row, the 403rd row) is enhanced, preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 67% and 162% or higher between.

In one embodiment, under the situation that the activity of e. coli k12 protein b1926 or flagellin fliT or its homologue (shown in Table II the 5th or 7 row, the 404th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 7% and 27% or higher between.

In one embodiment, in e. coli k12 protein b2307 or Histidine and Methionin/arginine/ornithine translocator (ATP-binding cassette superfamily, film) or under the activity situation about being enhanced of its homologue (shown in Table II the 5th or 7 row, the 405th and 406 row), preferably, give in one embodiment fine chemicals, preferred arginine be increased in 95% and 247% or more between, preferred L-glutamic acid be increased in 35% and 89% or more between, preferred arginine and L-glutamic acid be increased in 35% and 247% or higher between.

In one embodiment, (be listed as at e. coli k12 protein b2414 or its homologue as Table II the 5th or 7, the 407th the row shown in) activity be enhanced, for example threonine dehydra(ta)se superfamily activity of proteins is enhanced, the preferred amino acid bio that participates in synthesizes, halfcystine-aromatic series biosynthesizing, halfcystine-die aromatischen Aminosaeuren degraded, nitrogen and sulphur utilization, the aspartate family biosynthesizing, the aspartate family amino acid degradation, sulfuric acid and the biosynthesizing of L-cysteine derivative, biosynthesizing from elementary amino acid whose secondary species, from glycine, the biosynthesizing of the secondary species of L-Serine and L-L-Ala, pyridoxal phosphate bonded activity of proteins is enhanced, under the situation that the activity of preferred PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue is enhanced, preferably, give each fine chemicals, preferred glutamine be increased in 30% and 56% or higher between.

In one embodiment, activity at e. coli k12 protein b2426 or its homologue (shown in Table II the 5th or 7 row, the 408th row) is enhanced, for example have under the situation that the activity of the oxydo-reductase of NAD (P)-binding domains is enhanced, preferably, give each fine chemicals, preferred glutamine be increased in 31% and 62% or higher between.

In one embodiment, activity at e. coli k12 protein b2489 or its homologue (shown in Table II the 5th or 7 row, the 409th row) is enhanced, for example under the situation that the activity of hydrogenase Fe-S subunit is enhanced, preferably, give each fine chemicals, preferred glutamine be increased in 33% and 44% or higher between.

In one embodiment, activity at e. coli k12 protein b2553 or its homologue (shown in Table II the 5th or 7 row, the 410th and 411 row) is enhanced, for example under the situation that the activity of glutamine synthetase adjusting albumen P-II is enhanced, preferably, give each fine chemicals, preferred glutamine be increased in 55% and 90% or more between, preferred proline(Pro) be increased in 49% and 68% or more between, preferred glutamine and proline(Pro) be increased in 49% and 90% or higher between.

In one embodiment, activity at e. coli k12 protein b2644 or its homologue (shown in Table II the 5th or 7 row, the 412nd row) is enhanced, for example under the situation that the activity of hydrogenase Fe-S subunit is enhanced, preferably, give each fine chemicals, preferred proline(Pro) be increased in 35% and 853% or higher between.

At e. coli k12 protein b2710 or its homologue, for example flavine rubredoxin (FIRd) bi-functional NO and O ₂Under the activity situation about being enhanced of reductase enzyme (for example shown in Table II the 5th or 7 row, the 413rd row), preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 35% and 38% or higher between.

In one embodiment, activity at e. coli k12 protein b2818 or its homologue (shown in Table I the 5th or 7 row, the 414th and 415 row) is enhanced, for example under the situation that the activity of N-acetylglutamat synthase (amino acid N-Transacetylase) is enhanced, preferably, give each fine chemicals, preferred L-glutamic acid be increased in 50% and 129% or more between, preferred glutamine be increased in 45% and 519% or more between, preferred L-glutamic acid and glutamine be increased in 45% and 519% or higher between.

At e. coli k12 protein b3064 or its homologue, for example under the situation that the activity of Jia Ding the O-sialoglycoprotein endopeptidase with Actin muscle sample ATP enzymatic structure territory (for example shown in Table II the 5th or 7 row, the 416th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 72% and 141% or higher between.

Activity at e. coli k12 protein b3074 or its homologue (shown in Table II the 5th or 7 row, the 417th row) is enhanced, for example under the situation that the activity of tRNA synthetic enzyme is enhanced, preferably, give each fine chemicals, preferred L-glutamic acid be increased in 34% and 85% or higher between.

Activity at e. coli k12 protein b3116 or its homologue (shown in Table II the 5th or 7 row, the 418th row) is enhanced, for example under the situation that the activity of anaerobic inducibility L-Threonine/L-Serine permease (HAAAP family) is enhanced, preferably, give each fine chemicals, preferred L-glutamic acid be increased in 35% and 98% or higher between.

Activity at e. coli k12 protein b3160 or its homologue (shown in Table II the 5th or 7 row, the 419th row) is enhanced, for example have under the situation that the activity of the monooxygenase of luciferase sample atpase activity is enhanced, preferably, give each fine chemicals, preferred glutamine be increased in 38% and 189% or higher between.

At e. coli k12 protein b3166 or its homologue, for example under the activity situation about being enhanced of tRNA Pseudouridine 5S synthase (for example shown in Table II the 5th or 7 row, the 420th row), preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 29% and 40% or higher between.

At e. coli k12 protein b3169 or its homologue, for example under the activity situation about being enhanced of Transcription Termination-antitermination factor (for example shown in Table II the 5th or 7 row, the 421st and 422 row), preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 55% and 111% or more between, preferred L-glutamic acid be increased in 42% and 140% or more between, preferred glutamine and L-glutamic acid be increased in 42% and 140% or higher between.

At e. coli k12 protein b3231 or its homologue, for example under the activity situation about being enhanced of 50S ribosomal subunit protein matter L13 (for example shown in Table II the 5th or 7 row, the 423rd row), preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 50% and 164% or higher between.

At e. coli k12 protein b3619 or its homologue, for example under the activity situation about being enhanced of the ADP-L-glycerine of NAD (P)-associativity-D-mannoheptose-6-epimerase (for example shown in Table II the 5th or 7 row, the 424th row), preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 40% and 122% or higher between.

At e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 425th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 32% and 241% or higher between.

At e. coli k12 protein b3680 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 426th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 50% and 199% or higher between.

At e. coli k12 protein b3791 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 427th and 428 row) is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 28% and 57% or more between, preferred L-glutamic acid be increased in 39% and 57% or more between, preferred glutamine and L-glutamic acid be increased in 28% and 57% or higher between.

At e. coli k12 protein b3919 or its homologue, for example under the activity situation about being enhanced of triose-phosphate isomerase (for example shown in Table II the 5th or 7 row, the 429th row), preferably, give in one embodiment fine chemicals, preferred proline(Pro) be increased in 35% and 118% or higher between.

At e. coli k12 protein b3936 or its homologue, for example under the activity situation about being enhanced of 50S ribosomal subunit protein matter L32 (for example shown in Table II the 5th or 7 row, the 430th row), preferably, give in one embodiment fine chemicals, preferred arginine be increased in 120% and 398% or higher between.

At e. coli k12 protein b4004 or its homologue, for example under the activity situation about being enhanced of transcription regulatory protein (for example shown in Table II the 5th or 7 row, the 431st row), preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 30% and 36% or higher between.

At e. coli k12 protein b4074 or its homologue, for example under the situation that the biological activity that albumen (for example shown in Table II the 5th or 7 row, the 432nd row) takes place of cytochrome c-type is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 40% and 42% or higher between.

At e. coli k12 protein b4133 or its homologue, under the situation that the activity of for example pH transcriptional activation agent (OmpR family) of replying (for example shown in Table II the 5th or 7 row, the 433rd row) is enhanced, preferably, give in one embodiment fine chemicals, preferred glutamine be increased in 59% and 212% or higher between.

At e. coli k12 protein b4346 or its homologue, for example under the activity situation about being enhanced of 5-methylcytosine specificity restriction enzyme McrBC composition (for example shown in Table II the 5th or 7 row, the 434th row), preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 38% and 44% or higher between.

At yeast saccharomyces cerevisiae protein YFL019C or its homologue, for example have under the activity situation about being enhanced of active protein shown in [0022.0.4.4] (for example shown in Table II the 5th or 7 row, the 435th row), preferably, give in one embodiment fine chemicals, preferred L-glutamic acid be increased in 81% and 134% or higher between.

At e. coli k12 protein b0695 or its homologue (shown in Table I the 5th or 7 row, the 35th row), for example under the situation that the activity of sensibility histidine kinase is enhanced, preferably giving fine chemicals and phenylalanine increases [0046.0.4.4].

At e. coli k12 protein b0730 or its homologue (shown in Table I the 5th or 7 row, the 43rd or 54 row), for example under the situation that the activity of transcriptional is enhanced, preferably giving fine chemicals and fumaric acid increases.

(as directed Table I the 5th or 7 row, the 36th row, for example under the situation that the activity of transcriptional is enhanced, preferably giving fine chemicals and fumaric acid increases at e. coli k12 protein b1284 or its homologue.

At e. coli k12 protein b1827 or its homologue (shown in Table I the 5th or 7 row, the 56th row), for example under the situation that the activity of transcription repressor is enhanced, preferably giving fine chemicals and Isoleucine increases.

Activity at e. coli k12 protein b1829 or its homologue (shown in Table I the 5th or 7 row, the 34th or 60 row) is enhanced, for example have under the situation that the activity of the heat shock protein (htpx) of protease activity is enhanced, preferably giving fine chemicals and Isoleucine increases.

Activity at e. coli k12 protein b1852 or its homologue (shown in Table I the 5th or 7 row, the 61st row) is enhanced, and for example under the situation that the activity of glucose-6-phosphate dehydrogenase (G6PD) is enhanced, preferably giving fine chemicals and inositol increases.

Activity at e. coli k12 protein b2095 or its homologue (shown in Table I the 5th or 7 row, the 37th row) is enhanced, and for example under the situation that the activity of tagatose-6-phosphokinase is enhanced, preferably giving fine chemicals and L-Ala increases.

Activity at e. coli k12 protein b2699 or its homologue (shown in Table I the 5th or 7 row, the 55th row) is enhanced, and for example under the situation that the activity of recombinant protein recA is enhanced, preferably giving fine chemicals and fumaric acid increases.

At yeast saccharomyces cerevisiae protein YFR042W or its homologue (shown in Table I the 5th or 7 row, the 58th row), for example under the situation that the activity of " protein that the yeast cell survival is required " is enhanced, preferably giving fine chemicals and leucine increases.

At yeast saccharomyces cerevisiae protein YHR130C or its homologue (shown in Table I the 5th or 7 row, the 31st row), for example under the situation that the activity of " not profiling protein matter YHR130C " is enhanced, preferably giving fine chemicals and phenylalanine increases.

At yeast saccharomyces cerevisiae protein YIL150C or its homologue (shown in Table I the 5th or 7 row, the 51st row), for example under the situation that the activity of chromobindins is enhanced, preferably giving fine chemicals and Xie Ansuan increases.

At yeast saccharomyces cerevisiae protein YPR024W or its homologue (shown in Table I the 5th or 7 row, the 41st row), for example under the situation that the activity of the mitochondrial protein of the ATP of CDC48/PAS1/SEC18 family enzyme is enhanced, preferably giving fine chemicals and fumaric acid increases.

At yeast saccharomyces cerevisiae protein YPR138C or its homologue (shown in Table I the 5th or 7 row, the 53rd row), for example under the situation that the activity of ammonium transporter is enhanced, preferably giving fine chemicals and phenylalanine increases.

At yeast saccharomyces cerevisiae protein YBR204C or its homologue (shown in Table I the 5th or 7 row, the 38th row), for example under the situation that the activity of peroxysome lipase is enhanced, preferably giving fine chemicals and inositol increases.

At yeast saccharomyces cerevisiae protein YDR271C or its homologue (shown in Table I the 5th or 7 row, the 46th row), for example under the situation that the activity of " not profiling protein matter YDR271C " is enhanced, preferably giving fine chemicals and Isoleucine increases.

At yeast saccharomyces cerevisiae protein YER173W or its homologue (shown in Table I the 5th or 7 row, the 48th or 57 row), for example under the situation that the check position activity of proteins is enhanced, preferably giving fine chemicals and Xie Ansuan increases.

At yeast saccharomyces cerevisiae protein YFL013C or its homologue (shown in Table I the 5th or 7 row, the 39th row), for example under the situation that the activity of " INO80 chromatin reconstitution complex subunit " is enhanced, preferably giving fine chemicals and Xie Ansuan increases.

At yeast saccharomyces cerevisiae protein YFL050C or its homologue (shown in Table I the 5th or 7 row, the 49th row), for example under the situation that the activity of two-trivalent inorganic cation transporter is enhanced, preferably giving fine chemicals and Threonine increases.

At yeast saccharomyces cerevisiae protein YGR104C or its homologue (shown in Table I the 5th or 7 row, the 40th row), for example under the situation that the activity of " yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 " is enhanced, preferably giving fine chemicals and Isoleucine increases.

At yeast saccharomyces cerevisiae protein YKR057W or its homologue (shown in Table I the 5th or 7 row, the 32nd or 59 row), for example be similar under the situation that the activity of the ribosomal protein of S21A, S26A and/or YS25 ribosomal protein is enhanced, preferably giving fine chemicals and Threonine increases.

At e. coli k12 protein b0050 or its homologue, under the activity situation about being enhanced of the conservative protein matter of for example potential participation protein-protein interaction (for example shown in Table II the 5th or 7 row, the 386th row), preferably, give fine chemicals, preferred L-glutamic acid and another amino acid or more amino acids increase in one embodiment.

At e. coli k12 protein b0057 or its homologue, for example have such as [0022.0.4.4] under the activity situation about being enhanced of the active protein of definition (for example shown in Table II the 5th or 7 row, the 387th row), preferably, give fine chemicals, preferred L-glutamic acid and another amino acid or more amino acids increase in one embodiment.

At e. coli k12 protein b0138 or its homologue, for example under the activity situation about being enhanced of pili sample adhesin protein (for example shown in Table II the 5th or 7 row, the 388th row), preferably, giving fine chemicals, preferred proline(Pro) and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b0149 or its homologue, bi-functional penicillin-conjugated protein 1b for example: glycosyltransferase (N-end); Under the activity situation about being enhanced of transpeptidase (C-end) (for example shown in Table II the 5th or 7 row, the 389th row), preferably, giving fine chemicals, preferred proline(Pro) and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b0161 or its homologue, for example under the activity situation about being enhanced of pericentral siphon serine protease (for example as Table II the 5th or 7 row, 390-392 are capable shown in), preferably, giving fine chemicals, preferred arginine and/or L-glutamic acid and/or glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b0486 or its homologue, for example under the activity situation about being enhanced of amino acid/amine transporter (APC family) (for example shown in Table II the 5th or 7 row, the 393rd row), preferably, giving each fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b0849 or its homologue, for example under the activity situation about being enhanced of the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase (for example shown in Table II the 5th or 7 row, the 394th row), preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b0970 or its homologue, for example under the activity situation about being enhanced of glutamate receptor (for example shown in Table II the 5th or 7 row, the 395th row), preferably, giving each fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

In one embodiment, activity at e. coli k12 protein b1343 or its homologue (shown in Table II the 5th or 7 row, the 396th and 397 row) is enhanced, the activity of proteins that for example participates in rRNA processing and/or translation is enhanced, preferably under the situation that the activity of the ATP RNA-dependent helicase that is stimulated by 23SrRNA or its homologue is enhanced, preferably, giving each fine chemicals, preferred glutamine and/or glutamic acid and one or more other amino acid increases.

At e. coli k12 protein b1360 or active protein or its homologue with dna replication protein matter of the supposition of being defined as, for example under the activity situation about being enhanced of transcriptional (for example shown in Table II the 5th or 7 row, the 398th row), preferably, giving fine chemicals, preferred proline(Pro) and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b1693 or its homologue, for example under the activity situation about being enhanced of 3-dehydroquinate dehydratase (for example shown in Table II the 5th or 7 row, the 399th row), preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b1736 or its homologue, for example under the activity situation about being enhanced of PEP dependency phosphotransferase (for example shown in Table II the 5th or 7 row, the 400th row), preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

In one embodiment, at e. coli k12 protein b1738 or have under the situation that the activity of the active protein that is defined as PEP dependency phosphotransferase or its homologue (shown in Table II the 5th or 7 row, the 401st row) is enhanced, preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

In one embodiment, at e. coli k12 protein b1886 or methyl acceptor chemotactic protein II, under the activity situation about being enhanced of aspartic acid transmitter acceptor or its homologue (shown in Table II the 5th or 7 row, the 402nd row), preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

In one embodiment, under the situation that the activity of e. coli k12 protein b1896 or trehalose-6-phosphate synthase or its homologue (shown in Table II the 5th or 7 row, the 403rd row) is enhanced, preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

In one embodiment, under the situation that the activity of e. coli k12 protein b1926 or flagellin fliT or its homologue (shown in Table II the 5th or 7 row, the 404th row) is enhanced, preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

In one embodiment, under the situation that the activity of e. coli k12 protein b2307 or flagellin fliT or its homologue (shown in Table II the 5th or 7 row, the 405th and 406 row) is enhanced, preferably, giving fine chemicals, preferred arginine and/or L-glutamic acid and one or more other amino acid in one embodiment increases.

In one embodiment, (be listed as at e. coli k12 protein b2414 or its homologue as Table II the 5th or 7, the 407th the row shown in) activity be enhanced, for example threonine dehydra(ta)se superfamily activity of proteins is enhanced, the preferred amino acid bio that participates in synthesizes, halfcystine-aromatic series biosynthesizing, halfcystine-die aromatischen Aminosaeuren degraded, nitrogen and sulphur utilization, the aspartate family biosynthesizing, the aspartate family amino acid degradation, sulfuric acid and the biosynthesizing of L-cysteine derivative, biosynthesizing from elementary amino acid whose secondary species, from glycine, the biosynthesizing of the secondary species of L-Serine and L-L-Ala, pyridoxal phosphate bonded activity of proteins is enhanced, under the situation that the activity of preferred PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue is enhanced, preferably, give each fine chemicals, preferred glutamine and one or more other amino acid increase.

In one embodiment, activity at e. coli k12 protein b2426 or its homologue (shown in Table II the 5th or 7 row, the 408th row) is enhanced, for example have under the situation that the activity of the oxydo-reductase of NAD (P)-binding domains is enhanced, preferably, giving each fine chemicals, preferred glutamine and one or more other amino acid increases.

In one embodiment, activity at e. coli k12 protein b2489 or its homologue (shown in Table II the 5th or 7 row, the 409th row) is enhanced, for example under the situation that the activity of hydrogenase Fe-S subunit is enhanced, preferably, giving each fine chemicals, preferred glutamine and one or more other amino acid increases.

In one embodiment, activity at e. coli k12 protein b2553 or its homologue (shown in Table II the 5th or 7 row, the 410th and 411 row) is enhanced, for example under the situation that the activity of glutamine synthetase adjusting albumen P-II is enhanced, preferably, giving each fine chemicals, preferred glutamine and/or proline(Pro) and one or more other amino acid increases.

In one embodiment, activity at e. coli k12 protein b2644 or its homologue (shown in Table II the 5th or 7 row, the 412nd row) is enhanced, for example under the situation that the activity of hydrogenase Fe-S subunit is enhanced, preferably, giving each fine chemicals, preferred proline(Pro) and one or more other amino acid increases.

At e. coli k12 protein b2710 or its homologue, for example flavine rubredoxin (FIRd) bi-functional NO and O ₂Under the activity situation about being enhanced of reductase enzyme (for example shown in Table II the 5th or 7 row, the 413rd row), preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

In one embodiment, activity at e. coli k12 protein b2818 or its homologue (shown in Table I the 5th or 7 row, the 414th and 415 row) is enhanced, for example under the situation that the activity of N-acetylglutamat synthase (amino acid N-Transacetylase) is enhanced, preferably, giving each fine chemicals, preferred L-glutamic acid and/or glutamine and one or more other amino acid increases.

At e. coli k12 protein b3064 or its homologue, for example under the activity situation about being enhanced of Jia Ding O-sialoglycoprotein endopeptidase (for example shown in Table II the 5th or 7 row, the 416th row) with Actin muscle sample ATP enzymatic structure territory, preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

Activity at e. coli k12 protein b3074 or its homologue (shown in Table II the 5th or 7 row, the 417th row) is enhanced, for example under the situation that the activity of tRNA synthetic enzyme is enhanced, preferably, giving each fine chemicals, preferred L-glutamic acid and one or more other amino acid increases.

Activity at e. coli k12 protein b3116 or its homologue (shown in Table II the 5th or 7 row, the 418th row) is enhanced, for example under the situation that the activity of anaerobic inducibility L-Threonine/L-Serine permease (HAAAP family) is enhanced, preferably, giving each fine chemicals, preferred L-glutamic acid and one or more other amino acid increases.

Activity at e. coli k12 protein b3160 or its homologue (shown in Table II the 5th or 7 row, the 419th row) is enhanced, for example have under the situation that the activity of the monooxygenase of luciferase sample atpase activity is enhanced, preferably, giving each fine chemicals, preferred glutamine and one or more other amino acid increases.

At e. coli k12 protein b3166 or its homologue, for example under the activity situation about being enhanced of tRNA Pseudouridine 5S synthase (for example shown in Table II the 5th or 7 row, the 420th row), preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3169 or its homologue, for example under the activity situation about being enhanced of Transcription Termination-antitermination factor (for example shown in Table II the 5th or 7 row, the 421st and 422 row), preferably, giving fine chemicals, preferred glutamine and/or glutamic acid and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3231 or its homologue, for example under the activity situation about being enhanced of 50S ribosomal subunit protein matter L13 (for example shown in Table II the 5th or 7 row, the 423rd row), preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3619 or its homologue, for example under the activity situation about being enhanced of the ADP-L-glycerine of NAD (P)-associativity-D-mannoheptose-6-epimerase (for example shown in Table II the 5th or 7 row, the 424th row), preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 425th row) is enhanced, preferably, giving fine chemicals, preferred proline(Pro) and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3680 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 426th row) is enhanced, preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3791 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 427th and 428 row) is enhanced, preferably, giving fine chemicals, preferred glutamine and/or glutamic acid and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3919 or its homologue, for example under the activity situation about being enhanced of triose-phosphate isomerase (for example shown in Table II the 5th or 7 row, the 429th row), preferably, giving fine chemicals, preferred proline(Pro) and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b3936 or its homologue, for example under the activity situation about being enhanced of 50S ribosomal subunit protein matter L32 (for example shown in Table II the 5th or 7 row, the 430th row), preferably, giving fine chemicals, preferred arginine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b4004 or its homologue, for example under the activity situation about being enhanced of transcription regulatory protein (for example shown in Table II the 5th or 7 row, the 431st row), preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b4074 or its homologue, for example under the situation that the biological activity that albumen (for example shown in Table II the 5th or 7 row, the 432nd row) takes place of cytochrome c-type is enhanced, preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b4133 or its homologue, under the situation that the activity of for example pH transcriptional activation agent (OmpR family) of replying (for example shown in Table II the 5th or 7 row, the 433rd row) is enhanced, preferably, giving fine chemicals, preferred glutamine and one or more other amino acid in one embodiment increases.

At e. coli k12 protein b4346 or its homologue, for example under the activity situation about being enhanced of 5-methylcytosine specificity restriction enzyme McrBC composition (for example shown in Table II the 5th or 7 row, the 434th row), preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

At yeast saccharomyces cerevisiae protein YFL019C or its homologue, for example have under the activity situation about being enhanced of active protein shown in [0022.0.4.4] (for example shown in Table II the 5th or 7 row, the 435th row), preferably, giving fine chemicals, preferred L-glutamic acid and one or more other amino acid in one embodiment increases.

[0047.0.0.4] and [0048.0.0.4]: see [0047.0.0.0] and [0048.0.0.0]

[0049.0.4.4] has to give and improves arginine quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, 30-37,390, the polypeptide of consensus sequence shown in 405 and/or 430 row, perhaps as Table II the 5th or 7 row, 30-37,390, its function homologue described in polypeptide or the literary composition shown in 405 and/or 430 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, 30-37,390, nucleic acid molecule or its function homologue as herein described shown in 405 and/or 430 row) structure of coded polypeptide, and have the activity described in the literary composition.

Have to give and improve L-glutamic acid quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, 38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427, the polypeptide of consensus sequence shown in 434 and/or 435 row, perhaps as Table II the 5th or 7 row, 38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427, its function homologue described in polypeptide or the literary composition shown in 434 and/or 435 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, 38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427, nucleic acid molecule or its function homologue as herein described shown in 434 and/or 435 row) structure of coded polypeptide, and have the activity described in the literary composition.

Have to give and improve proline(Pro) quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, 44-56,388,389,398,411,412, the polypeptide of consensus sequence shown in 425 and/or 429 row, perhaps as Table II the 5th or 7 row, 44-56,388,389,398,411,412, its function homologue described in polypeptide or the literary composition shown in 425 and/or 429 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, 44-56,388,389,398,411,412, nucleic acid molecule or its function homologue as herein described shown in 425 and/or 429 row) structure of coded polypeptide, and have the activity described in the literary composition.

Have to give and improve glutamine quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, 57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide of consensus sequence, perhaps as Table II the 5th or 7 row, 57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in its function homologue described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, 57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in nucleic acid molecule or its function homologue as herein described) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.4.4] for the purposes of the present invention, term " arginine " and/or " L-glutamic acid " and/or " glutamine " and/or " proline(Pro) " and " L-arginine " and/or " L-L-glutamic acid " and/or " L-glutaminate " and/or " L-proline(Pro) " also comprise corresponding salt, for example, the vitriol of the hydrochloride of arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro) or arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro).Preferably, term arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro) are intended to comprise term L-arginine and/or L-L-glutamic acid and/or L-glutaminate and/or L-proline(Pro).

[0051.0.0.4] and [0052.0.0.4]: see [0051.0.0.0] and [0052.0.0.0]

[0053.0.4.4] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention (for example has as Table II the 3rd row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide) express to increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention and (are for example had as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide) express to improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or the of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide) express to improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by the coded protein of nucleic acid molecule of the present invention, polypeptide perhaps of the present invention (for example has as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide) express to improve;

(e) by add the activity that one or more external source inducible factors come stimulating protein in biological or its part, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide) express to improve;

(f) transgenosis of expression coded protein, described protein is given the activity with each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide) express to improve; And/or

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, and described nucleic acid molecule encoding has the active of each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide);

(h) by adding positive Expression element or removing negative Expression element and improve code book invention polypeptide and (for example have as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or remove straining element from control region.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.4.4] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having according to Table II 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the capable protein of 431-433 or its homologue (for example Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide) give arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine after active expression of polypeptides or the activity and increase.

[0055.0.0.4] to [0071.0.0.4]: see that [0055.0.0.0] is to [0071.0.0.0]

[0072.0.4.4] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has argininosuccinic acid, citrulline, ornithine, urea, dihydropyrrol-5-carboxylic acid, oxyproline, hydroxyl dihydropyrrole-5-carboxylate, 3-hydroxyl dihydropyrrol-5-carboxylic acid, gamma-glutamyl cysteine, gsh, hydroxygultamic acid, 4-hydroxygultamic acid, keto-glutaric acid, 4-hydroxyl-2-oxopentanedioic acid, glutamine except arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro).

[0073.0.4.4] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) (for example Table II the 5th or 7 is listed as to improve polypeptide of the present invention or its homologue, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, for example give biology (preferred microorganism, the non-human animal, plant or zooblast, plant or animal tissues or plant) in each fine chemicals improve;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or bonded fine chemicals by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation, and optional other dissociates and/or in conjunction with amino acid.

[0074.0.0.4] to [0084.0.0.4]: see that [0074.0.0.0] is to [0084.0.0.0]

[0085.0.4.4] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the genetic regulatory element that effectively connects of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.4] and [0088.1.0.4]: see [0086.0.0.0] and [0088.1.0.0]

[0089.0.0.4] to [0097.0.0.4]: see that [0089.0.0.0] is to [0097.0.0.0]

[0098.0.4.4] in preferred embodiments, fine chemicals (arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro)) be produce according to the present invention and carry out isolating where necessary.The aminoacid mixture that the method according to this invention produces other amino acid such as methionine(Met), Methionin and/or Threonine is favourable.

[0099.0.0.4] to [0102.0.0.4]: see that [0099.0.0.0] is to [0102.0.0.0]

[0103.0.4.4] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide has as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence, its quantity of giving each fine chemicals in biology or its part increases;

(b) the preferred nucleic acid molecule that comprises the mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has as Table III the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprises and has as Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.0.4.] is used for nucleic acid molecule and the Table I A the 5th or 7 row of the inventive method in one embodiment, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the nucleic acid molecule that is used for the inventive method is not by Table I A the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence form.In one embodiment, the nucleic acid molecule and Table I A the 5th or 7 row that are used for the inventive method, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide of sequence.

[00103.2.0.4.] is used for nucleic acid molecule and the Table I B the 5th or 7 row of the inventive method in one embodiment, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the nucleic acid molecule that is used for the inventive method is not by Table I B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence form.In one embodiment, the nucleic acid molecule and Table I B the 5th or 7 row that are used for the inventive method, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide of sequence.

[0104.0.4.4] is used for nucleic acid molecule and the Table I the 5th or 7 row of the inventive method in one embodiment, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the nucleic acid that is used for the inventive method not by Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide of sequence.

[0105.0.0.4] to [0107.0.0.4]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.4.4] method of the present invention and had as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleic acid molecule of sequence, from being listed as Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in aminoacid sequence derive or from containing just like Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide deutero-nucleic acid molecule of consensus sequence, perhaps its coding has as Table I the 3rd, 5 or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the active or bioactive polypeptide of polypeptidase or for example give derivative or the homologue that fine chemicals increases behind its expression or active the increasing.

[0109.0.0.4]: see [0109.0.0.0]

[0110.0.4.4] helps the inventive method and encodes to have the polypeptide of polypeptide of the present invention or the used polypeptide active of the used or of the present invention process of the inventive method, for example as Table II the 5th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or by as Table I the 5th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the protein of nucleic acid molecule encoding or its homologue (Table II the 7th row for example, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) nucleic acid molecule can from generally open database, determine.

[0111.0.0.4]: see [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.4.4] the inventive method is the isolated nucleic acid sequences form, its coding has as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide of protein active, and give arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine and increase.

[0113.0.0.4] to [0120.0.0.4]: [0113.0.0.0] is to [0120.0.0.0]

[0121.0.4.4] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in peptide sequence or its functional homologue as herein described the difference of one or more amino acid moleculars is arranged, described artificial sequence is preferably given aforementioned activity, and promptly giving each fine chemicals after improving its activity increases.

[0122.0.0.4] to [0127.0.0.4]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.4.4] polymerase chain reaction (PCR) amplification is used is (for example as Table III the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the primer of 431-433 shown in capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable or from as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence deutero-sequence produce.

[0129.0.4.4] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of the inventive method (the particularly sequence of polypeptide of the present invention).Indicate in the shown in the drawings comparison of the conservative region of polypeptide of the present invention.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in consensus sequence from described comparison.

[0130.0.0.4] to [0138.0.0.4]: see that [0130.0.0.0] is to [0138.0.0.0]

[0139.0.4.4] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna is listed as with Table I the 5th or 7 under loose hybridization conditions, among the preferred Table I B for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence hybridization, and coding is expressed and to be had each fine chemicals of increasing, the active peptide of arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine particularly.

[0140.0.0.4] to [0146.0.0.4]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.4.4] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: one of it and described nucleotide sequence are fully complementary, thereby can and form stable duplex with the hybridization of one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.4.4] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 34-37 among the preferred Table I B, 390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419 funtion part homologys are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine.

[0149.0.4.4] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the capable resp. of 431-433, preferred of Table I B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in one of nucleotide sequence or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and protein as shown in Table II.

[00149.1.4.4] randomly, with Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table I B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd be listed as, the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the protein of 431-433 shown in capable be known activity or be used for these protein of note.

[0150.0.4.4] in addition, nucleic acid molecule of the present invention can only contain Table I the 5th or 7 row, preferred Table I B 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and described nucleotides sequence column region is listed as with Table I the 5th or 7 under stringent condition, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the sense strand of one of sequence, Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotide hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the right PCR of primer will produce as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in fragment or its gene product of polynucleotide sequence.

[0151.0.0.4]: see [0151.0.0.0]

[0152.0.4.4] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and are listed as Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising arginine as be shown in the examples and/or L-glutamic acid and/or proline(Pro) and/or glutamine.

[0153.0.4.4] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprises and is listed as Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), so that this protein or its part can participate in increasing the generation of each fine chemicals.In one embodiment, as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein or its part have for example Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the activity of polypeptide.

[0154.0.4.4] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% also most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.4] and [0156.0.0.4]: see [0155.0.0.0] and [0156.0.0.0]

[0157.0.4.4] the present invention relates in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in consensus sequence polypeptide or as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the consensus sequence of 431-433 shown in capable or as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the consensus sequence of 431-433 shown in capable or as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet, in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable, preferably as Table I A the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in nucleic acid molecule identical.

[0158.0.0.4] to [0160.0.0.4]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.4.4] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it (for example comprises Table I the 5th or 7 row with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used under stringent condition, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleic acid molecule of sequence) hybridization.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.4]: see [0162.0.0.0]

[0163.0.4.4] preferably is listed as with Table I the 5th or 7 under stringent condition, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.4]: see [0164.0.0.0]

[0165.0.4.4] for example can (for example be listed as Table I the 5th or 7 in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence) in produce the Nucleotide that the amino acid cause " nonessential " amino-acid residue place replaces and replace.

[0166.0.0.4] and [0167.0.0.4]: see [0166.0.0.0] and [0167.0.0.0]

[0168.0.4.4] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the contained sequence difference of sequence, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contains and is listed as Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the aminoacid sequence of 431-433 shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or one of the sequence of 431-433 shown in capable identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.4] to [0172.0.0.4]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.4.4] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:1982 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:1982 sequence before use.

[0174.0.0.4]: see [0174.0.0.0]

[0175.0.4.4] for example, the sequence that has 80% homology at protein level and SEQ ID NO:1983 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQID NO:1983 sequence.

[0176.0.4.4] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the functional equivalent that obtains of one of polypeptide be listed as according to of the present invention as Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide have essentially identical character and discern.

[0177.0.4.4] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table I B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the functional equivalent that obtains of nucleotide sequence be listed as according to of the present invention as Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table I B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide of the essentially identical character of polypeptide.

[0178.0.0.4]: see [0178.0.0.0]

[0179.0.4.4] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in) in introduce the replacement of one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable, preferred Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to being listed as Table I the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 introduce sudden change in the encoding sequence of sequence shown in capable.

[0180.0.0.4] to [0183.0.0.4]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.4.4] is employed to have as Table I, preferred Table I B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleotide sequence homologous compound of sequence, perhaps come Table II the 5th or 7 row freely, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the homologous compound of nucleotide sequence of sequence also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence or from the deutero-nucleotide sequence) by the disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.4.4] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I, preferred Table I B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable.In one embodiment, the preferred nucleic acid molecule comprises the least possible, as Table I, preferred Table I B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the arbitrary sequence of 431-433 shown in capable in other Nucleotide of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable identical.

The employed one or more nucleic acid molecule encodings of [0186.0.4.4] also preferred the inventive method comprise as Table II, preferred Table II B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, be used for the encoded polypeptide of the inventive method with as Table II, preferred Table II B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence identical.

[0187.0.4.4] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprise as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II, preferred Table II B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the encoding sequence of sequence identical.

[0188.0.4.4] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or enzymic activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in and under the same conditions the polypeptide expressed activity compare and basic do not reduce.

In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence or by its homologue of forming.

[0189.0.4.4] is as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the homologous compound of sequence, or deutero-such as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the homologue of the sequence of 431-433 shown in capable also refer to truncated sequence, cDNA, the single stranded DNA or the RNA of coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.4] to [0203.0.0.4]: see that [0190.0.0.0] is to [0203.0.0.0]

[0204.0.4.4] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II, preferred Table II B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or polypeptide or its segmental nucleic acid molecule of 431-433 shown in capable, described nucleic acid molecule is given each fine chemicals in biological or its part, arginine (30-37 particularly, 390,405 and/or 430 go) and/or L-glutamic acid (38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 go) and/or proline(Pro) (44-56,388,389,398,411,412,425 and/or 429 go) and/or glutamine (57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable) quantity increases;

(b) comprise, preferably comprise being listed as of mature form at least as Table I the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or nucleic acid molecule or its segmental nucleic acid molecule of 431-433 shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the primer of 431-433 shown in capable or primer nucleic acid molecule that amplification from cDNA library or genomic library is obtained, and in biological or its part, give each fine chemicals, arginine (30-37 particularly, 390,405 and/or 430 go) and/or L-glutamic acid (38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row/or proline(Pro) (44-56,388,389,398,411,412,425 and/or 429 go) and/or glutamine (57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable) increase of quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like Table IV the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in consensus sequence and in biological or its part, give each fine chemicals, arginine (30-37 particularly, 390,405 and/or 430 go) and/or L-glutamic acid (38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 go) and/or proline(Pro) (44-56,388,389,398,411,412,425 and/or 429 go) and/or glutamine (57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable) nucleic acid molecule that increases of quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, arginine (30-37 particularly, 390,405 and/or 430 go) and/or L-glutamic acid (38-43,386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 go) and/or proline(Pro) (44-56,388,389,398,411,412,425 and/or 429 go) and/or glutamine (57-62,392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable) nucleic acid molecule that increases of quantity, described peptide coding such as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the structural domain of the polypeptide of 431-433 shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the nucleic acid molecule of 431-433 shown in capable or coding (optimized encoding is mature form at least) as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the 15nt at least of nucleic acid molecule of polypeptide, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as Table I A the 5th or 7 row by one or more Nucleotide, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable.In one embodiment, nucleic acid molecule not by Table I A or IB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable form.In one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable less than 100%, 99.999%, 99.99%, 99.9% or 99% is identical.In another embodiment, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide of sequence.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence at least 30%, 40%, 50% or 60% is identical and less than 100%, 99.999%, 99.99%, 99.9% or 99% is identical.In another embodiment, nucleic acid molecule is not encoded as Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the peptide sequence of 431-433 shown in capable.Therefore, in one embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in nucleic acid molecule have one or more residue differences at least.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the polypeptide of 431-433 shown in capable at least one or the different polypeptide of a plurality of amino acid.In another embodiment, Table I A or IB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the protein sequence of 431-433 shown in capable.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) be not by as Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in protein sequence at least 30%, 40%, 50% or 60% identical and with Table I A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% is identical.

[0205.0.0.4] to [0226.0.0.4]: see that [0205.0.0.0] is to [0226.0.0.0]

[0227.0.4.4] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in beyond the sequence or derivatives thereof, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example L-Methionin of at least one amino acid, other genes of L-Threonine and/or L-methionine(Met) and/or L-leucine and/or Isoleucine and/or Xie Ansuan biosynthetic pathway of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.4] to [0230.0.0.4]: see that [0228.0.0.0] is to [0230.0.0.0]

[0231.0.4.4] is in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro) simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.4] to [0282.0.0.4]: see that [0232.0.0.0] is to [0282.0.0.0]

[0283.0.4.4] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in proteinic antibody, perhaps anti-as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the antibody of polypeptide, it can utilize polypeptide of the present invention or its fragment (being polypeptide of the present invention) to produce by standard technique.Monoclonal antibody preferably.

[0284.0.0.4]: see [0284.0.0.0].

[0285.0.4.4] the present invention relates to have as Table II the 5th or 7 row in one embodiment, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence or by as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.4.4] improved in the inventive method and contained just like Table IV the 7th row in an advantageous embodiment, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in consensus sequence or by the activity of its polypeptide of forming.In another embodiment, the present invention relates to contain just like Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in consensus sequence or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise that the inventive method of polypeptide or relate to comprises more than one as Table IV the 7th row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide of (different rows) consensus sequence.

[0287.0.0.4] to [0290.0.0.4]: see that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.4.4] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as Table II A or IIB the 5th or 7 row by one or more amino acid, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence form.

[0292.0.0.4]: see [0292.0.0.0]

[0293.0.4.4] the present invention relates to give that each fine chemicals increases and by the polypeptide of nucleic acid molecule of the present invention or the employed nucleic acid molecule encoding of the inventive method in one embodiment in biological or its part.

In one embodiment, polypeptide of the present invention has by one or more amino acid and as Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in other sequence of sequence phase region.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence form.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide be can't help as Table I A or IB the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the sequence of nucleic acid molecule encoding form.

[0294.0.4.4] in one embodiment, the present invention relates to have as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide of activity of proteins, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II Aor Table II B the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the sequence of 431-433 shown in capable.

[0295.0.0.4] and [0297.0.0.4]: see [0295.0.0.0] and [0297.0.0.0]

The chemical of [0297.1.4.4] non-polypeptide of the present invention is for example not have Table II the 3rd, 5 or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the active polypeptide of polypeptide.

[0298.0.4.4] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as being listed as with Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the identical aminoacid sequence of sequence.

[0299.0.4.4] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in one of nucleotide sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and is listed as Table I the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.4.4] therefore, described in detail as this paper, polypeptide of the present invention is because natural variation or mutagenesis can be on aminoacid sequences and as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the sequence difference.Therefore, this polypeptide contains and is listed as Table II A or IIB the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.4]: see [0301.0.0.0]

The biologically-active moiety of [0302.0.4.4] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the aminoacid sequence of 431-433 shown in capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.4]: see [0303.0.0.0]

[0304.0.4.4] operation nucleic acid molecule of the present invention may cause that generation has basically as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide active and the protein that on sequence, there are differences with described wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered

[0305.0.0.4] to [0308.0.0.4]: see that [0306.0.0.0] is to [0308.0.0.0]

[0309.0.4.4] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 be meant the polypeptide that has corresponding polypeptide of the present invention or be used for the amino acid sequence of polypeptide of the inventive method shown in capable, be not listed as and be shown in Table II the 5th or 7,34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or " the non-of the present invention polypeptide " or " other polypeptide " of 431-433 in the capable polypeptide that is meant aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with as Table II the 5th or 7 be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide homology not basically, for example do not give described in the literary composition active or as Table II the 3rd be listed as, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in note or known and from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the increase of in organism or its part, not giving each fine chemicals of " the non-of the present invention polypeptide " or " other polypeptide " of 431-433 in capable.

[0310.0.0.4] to [0334.0.0.4]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.4.4] confirmed that the dsRNAi method is to reducing as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the expression of the nucleotide sequence of 431-433 shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates to when introducing in the biology, (or be derived from its cell in the time of in the preferred introduced plant, tissue, organ or seed), by reducing as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the expression of nucleotide sequence and/or its homologue and the double stranded rna molecule (dsRNA molecule) that causes metabolic activity to change.Be used for reducing as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of one of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.4] to [0342.0.0.4]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.4.4] is as describing, in order to cause effective reduction of expression, at dsRNA and as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example in a kind of organism as Table I the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.4] to [0361.0.0.4]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.4.4] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide, for example coding has as Table II the 3rd row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the polypeptide of protein active) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or nucleic acid molecule of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Have as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, be listed as Table II the 3rd in cell or biological or its part, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide (for example, have as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide of sequence) activity improve.Example and the inventive method are described in above.

[0363.0.0.4]: to [0382.0.0.4]: see that [0363.0.0.0] is to [0382.0.0.0]

[0383.0.4.4] in order to prepare fine chemicals, the particularly fine chemicals that contains arginine and/or L-glutamic acid and/or glutamine and/or proline compounds, can use organic compound for example citrulline, argininosuccinic acid, ornithine, aspartic acid, 2-oxopentanedioic acid, glutamy, glutamic semialdehyde, dihydropyrrol-5-carboxylic acid, glutamine or other organic arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro) precursor compound as the source of arginine and/or L-glutamic acid and/or glutamine and/or proline(Pro).

[0384.0.0.4]: see [0384.0.0.0]

The fermented liquid that particularly contains L-arginine for example and/or L-L-glutamic acid and/or L-proline(Pro) and/or L-tryptophane, L-methionine(Met), L-Threonine and/or L-Methionin that [0385.0.4.4] obtains with this kind approach, dry matter content accounts for 7.5 to 25% of weight usually.At least when finishing, particularly passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means the concentration that can utilize sugar in the fermention medium during this period of time remain on or be reduced to 〉=0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant) or these methods or partly from fermented liquid, shift out or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.0.4] to [0392.0.0.4]: see that [0386.0.0.0] is to [0392.0.0.0]

[0393.0.4.4] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(d) sample that will comprise candidate gene (be coded in and give the gene product that fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(e) evaluation and nucleic acid molecule of the present invention are particularly as Table I, preferred Table I B the 5th or 7 row, be respectively 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the nucleic acid molecule of 431-433 shown in the capable nucleic acid molecule of under lax stringent condition, hybridizing, and randomly separate full length cDNA clone or complete genome group clone;

(f) the candidate nucleic acid molecule is introduced the host cell that is fit to produce fine chemicals, preferred plant cell or microorganism;

(g) in host cell, express the nucleic acid molecule of being identified;

(h) level of fine chemicals in the analysis host cell; With

(i) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.4] to [0399.0.0.4]: see that [0394.0.0.0] is to [0399.0.0.0]

[00399.1.4.4]: it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect whether depend on as Table II the 5th or 7 row, be respectively 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the polypeptide of 431-433 shown in capable or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, be respectively 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.4] to [0423.0.0.4]: see that [0400.0.0.0] is to [0423.0.0.0]

[0424.0.0.4]: see [0424.0.0.2]

[0425.0.0.4] to [0460.0.0.4]: see that [0425.0.0.0] is to [0460.0.0.0]

[0461.0.4.4] embodiment 10: clone SEQ ID NO:1982 is used for expressing plant

[0462.0.0.4]: see [0462.0.0.0]

[0463.0.4.4] passes through pcr amplification SEQ ID NO:1982 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.4] to [0466.0.0.4]: see that [0464.0.0.0] is to [0466.0.0.0]

[0467.0.4.4] selects following primer sequence for genes of SEQ ID NO:1982:

I) forward primer (SEQ ID No:2046)

atgaataacg?aacccttacg?tccc

Ii) reverse primer (SEQ ID No:2047)

ttacatatcc?tcatgaaatt?cttcaagt

[0468.0.0.4] to [0479.0.0.4]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.4.4] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:1982

[0481.0.0.4] to [0513.0.0.4]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.4.4] selects as another kind of, as people such as Geigenberger (Plant Cell ﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect amino acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??b0730	L-glutamic acid	??LC	??1.55	??2.15
??b0730	Proline(Pro)	??GC	??1.35	??3.72
					??b1829	Glutamine	??LC	??1.50	??1.68
??b1829	Arginine	??LC	??1.45	??12.41
					??b2699	Proline(Pro)	??GC+LC	??1.32	??2.41
??YBR030W	Proline(Pro)	??LC	??1.51	??3.82
					??YBR204C	L-glutamic acid	??GC	??1.55	??1.76
??YDL106C	Proline(Pro)	??GC+LC	??1.51	??1.99
					??YDR271C	Proline(Pro)	??GC+LC	??1.36	??5.82
??YDR316W	Arginine	??LC	??1.45	??2.02
					??YEL045C	Proline(Pro)	??GC	??1.41	??1.89
??YER173W	Glutamine	??GC	??1.86	??3.85
					??YER173W	Proline(Pro)	??GC	??1.34	??2.91
??YFL013C	L-glutamic acid	??GC+LC	??1.81	??2.34
					??YFL050C	Proline(Pro)	??GC	??1.44	??1.74
??YFR042W	Glutamine	??GC	??1.41	??1.43
					??YGR104C	L-glutamic acid	??LC	??1.64	??1.96
??YGR135W	Proline(Pro)	??GC	??1.32	??3.89
					??YHR130C	Arginine	??LC	??1.67	??1.85
??YIL150C	Proline(Pro)	??GC	??1.33	??4.04
					??YKR057W	Arginine	??LC	??1.57	??5.57
??YKR057W	Glutamine	??GC	??1.41	??3.84
					??YNL090w	Proline(Pro)	??GC+LC	??1.73	??6.29
??YNL090w	Arginine	??LC	??1.54	??4.23

??YPR024W	L-glutamic acid	??LC	??1.26	??1.43
					??YPR133W-A	L-glutamic acid	??GC	??1.34	??1.68
??YPR138C	Proline(Pro)	??GC	??1.54	??6.20
					??b0695	Arginine	??LC	??1.51	??4.19
??b1284	Arginine	??LC	??1.47	??2.83
					??b1827	Proline(Pro)	??GC	??1.42	??2.26
??b1852	Glutamine	??GC	??1.40	??1.42
					??b2095	Arginine	??LC	??1.55	??1.59
??b4265	Glutamine	??GC	??1.32	??1.47
					??b0050	L-glutamic acid	??LC	??1.37	??1.97
??b0057	L-glutamic acid	??GC+LC	??1.35	??1.83
					??b0138	Proline(Pro)	??LC	??1.50	??2.80
??b0149	Proline(Pro)	??GC	??1.33	??2.20
					??b0161	Arginine	??LC	??7.28	??9.81
??b0161	L-glutamic acid	??LC	??1.35	??1.65
					??b0161	Glutamine	??GC	??1.43	??3.56
??b0486	Glutamine	??LC	??1.51	??2.28
					??b0849	Glutamine	??LC	??1.37	??1.50
??b0970	Glutamine	??GC+LC	??1.59	??3.80
					??b1343	Glutamine	??LC	??1.37	??1.39
??b1343	L-glutamic acid	??GC	??1.48	??1.99
					??b1360	Proline(Pro)	??GC	??1.33	??1.70
??b1693	L-glutamic acid	??LC	??1.39	??2.49
					??b1736	L-glutamic acid	??LC	??1.46	??1.97
??b1738	L-glutamic acid	??LC	??1.38	??2.07
					??b1886	Glutamine	??LC	??1.36	??2.24
??b1896	L-glutamic acid	??GC	??1.67	??2.62

??b1926	Glutamine	??LC	??1.07	??1.27
					??b2307	Arginine	??LC	??1.95	??3.47
??b2307	L-glutamic acid	??LC	??1.35	??1.89
					??b2414	Glutamine	??LC	??1.30	??1.56
??b2426	Glutamine	??LC	??1.31	??1.62
					??b2489	Glutamine	??LC	??1.33	??1.44
??b2553	Proline(Pro)	??LC	??1.49	??1.68
					??b2553	Glutamine	??GC+LC	??1.55	??1.90
??b2664	Proline(Pro)	??GC+LC	??1.35	??9.53
					??b2710	L-glutamic acid	??LC	??1.35	??1.38
??b2818	Glutamine	??GC	??1.45	??6.19
					??b2818	L-glutamic acid	??GC	??1.50	??2.29
??b3064	Glutamine	??GC	??1.72	??2.41
					??b3074	L-glutamic acid	??LC	??1.34	??1.85
??b3116	L-glutamic acid	??GC+LC	??1.35	??1.98
					??b3160	Glutamine	??LC	??1.38	??1.64
??b3160	Glutamine	??GC	??1.51	??2.89
					??b3166	Glutamine	??LC	??1.29	??1.40
??b3169	Glutamine	??GC+LC	??1.55	??2.11
					??b3169	L-glutamic acid	??GC+LC	??1.42	??2.40
??b3231	Glutamine	??GC	??1.50	??2.64
					??b3619	L-glutamic acid	??LC	??1.40	??2.22
??b3644	Proline(Pro)	??GC+LC	??1.32	??3.41
					??b3680	Glutamine	??GC	??1.50	??2.99
??b3791	Glutamine	??LC	??1.28	??1.57
					??b3791	L-glutamic acid	??LC	??1.39	??1.57
??b3919	Proline(Pro)	??GC	??1.35	??2.18

??b3936	Arginine	??LC	??2.20	??4.98
					??b4004	Glutamine	??LC	??1.30	??1.36
??b4074	Glutamine	??GC	??1.40	??1.42
					??b4133	Glutamine	??GC	??1.59	??3.12
??b4346	L-glutamic acid	??GC	??1.38	??1.44
					??YFL019C	L-glutamic acid	??GC+LC	??1.81	??2.34

[0515.0.0.4] to [0552.0.0.4]: see that [0515.0.0.0] is to [0552.0.0.0]

[0552.1.0.1]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF title	Metabolite	??Min	??Max
				??YKR057W	Glutamine	??2.30	??6.16
??YIL150C	Proline(Pro)	??1.77	??3.45

Table 2 shows that proline(Pro) and glutamine have increased in expressing the genetic modification maize plant of yeast saccharomyces cerevisiae nucleotide sequence YIL150c or YKR057w respectively.

In one embodiment, under the situation that yeast saccharomyces cerevisiae protein YIL150C or its homologue are enhanced as the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue in maize plant, preferably, giving the fine chemicals proline(Pro) is increased between 77% and 245%.

Under the situation that the activity of yeast saccharomyces cerevisiae protein YKR057W or the biological ribosomal protein that takes place and translate, be similar to the S21 ribosomal protein of participation rrna or its homologue is enhanced in maize plant, preferably, giving the fine chemicals glutamine is increased between 130% and 516%.

[0552.2.0.4]: see [0552.2.0.0]

[0553.0.4.4]

1. produce the method for arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine, it comprises:

(a) raising or generation are listed as Table II the 5th or 7 in non-human being or its one or more parts, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine.

2. produce the method for arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is as Table II the 5th or 7 row, this Wei be for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity to be increased;

B) contain just like Table I the 5th or 7 row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the nucleic acid molecule of the nucleic acid molecule of 431-433 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that arginine in biology or its part and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by using as Table III the 5th or 7 row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give in biology or its part arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity and increase;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively for arginic 34-37,390,405 and/or 430 the row and/or for the 43rd of L-glutamic acid, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or for the 54-56 of proline(Pro), 388,389,398,411,412,425 and/or 429 the row and/or for the 62nd of glutamine, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in consensus sequence polypeptide and give biology or its part in arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded arginine and/or L-glutamic acid and/or the proline(Pro) and/or the glutamine of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is as Table II the 5th or 7 row, 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity to be increased;

B) contain just like Table I the 5th or 7 row, be respectively 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or the nucleic acid molecule of the nucleic acid molecule of 431-433 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that arginine in biology or its part and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by using as Table III the 7th row, be respectively 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give in biology or its part arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity and increase;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in consensus sequence polypeptide and give biology or its part in arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, be respectively 34-37,390,405 and/or 430 the row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 the row and/or 54-56,388,389,398,411,412,425 and/or 429 the row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 9 or 10 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, it produces by the method described in claim 13 or by the nucleic acid molecule encoding described in claim 6, this polypeptide is different from as Table II A the 5th or 7 row by one or more amino acid thus, be respectively 34-37,390,405 and/or 430 row and/or the 43rd, 386,387,391,396,399-401,403,406,413,414,417,418,421,424,427,434 and/or 435 row and/or 54-56,388,389,398,411,412,425 and/or 429 row and/or the 62nd, 392-395,397,402,404,407-410,415,416,419,420,422,423,426,428 and/or 431-433 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the arginine that will measure and/or L-glutamic acid and/or proline(Pro) and/or glutamine level or expression of polypeptides level and the standard arginine of when described candidate compound or the sample that comprises described multiple compound lack, measuring and/or L-glutamic acid and/or proline(Pro) and/or glutamine or expression of polypeptides level; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that arginine in plant or the microorganism and/or L-glutamic acid and/or proline(Pro) and/or glutamine produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of expression of polypeptides of arginine in biology or its part and/or L-glutamic acid and/or proline(Pro) and/or the increase of glutamine quantity and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 5; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that arginine in the cell and/or L-glutamic acid and/or proline(Pro) and/or glutamine produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) arginine and/or L-glutamic acid and/or proline(Pro) and/or the glutamine level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give arginine and/or L-glutamic acid and/or proline(Pro) and/or the raising of glutamine level in the host cell after it is expressed with wild-type.

20. identify and to give the method that arginine in the cell and/or L-glutamic acid and/or proline(Pro) and/or glutamine produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that arginine in biology or its part and/or L-glutamic acid and/or proline(Pro) and/or glutamine quantity or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) arginine and/or L-glutamic acid and/or proline(Pro) and/or the glutamine level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give arginine and/or L-glutamic acid and/or proline(Pro) and/or the raising of glutamine level in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify the nucleic acid molecule of giving arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 14, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make anti-arginine and/or L-glutamic acid and/or proline(Pro) and/or the glutamine synthetic weedicide of suppressing of plant.

[0554.0.0.4] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.5] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.5] sees [0001.0.0.0] for disclosing of this section.

[0002.0.5.5] lipid acid is the building block of triacylglycerol, lipid, oils and fat.Some lipid acid, for example linolic acid or linolenic acid are " essential ", needed by human body is wanted because human body can not synthesize, and this time people must absorb by ingesting.Other lipid acid can be synthetic by human body, so they are not " essential ".However, health produce such as timnodonic acid (=EPA, C _20:5 ^{Δ 5,8,11,14,17}) or docosahexenoic acid (=DHA, C _22:6 ^{Δ 4,7,10,13,16,19}) amount common deficiency for best body function of lipid acid.Polyunsaturated fatty acid (=PUFA) referring to have on the carbochain more than one the lipid acid of two keys, its position according to the two keys of least significant end is divided into a plurality of families.The lipid acid that has two main hypotypes: ω-3 and ω-6 lipid acid.Omega-fatty acid is the lipid acid of least significant end double bond position on 3 carbon that begin from the methyl end.ω-6 lipid acid is the lipid acid of least significant end double bond position on 6 carbon that begin from the methyl end.Linolic acid (ω-6) and alpha-linolenic acid (ω-3) are unique real " essential " lipid acid.In vivo, these two kinds of lipid acid synthesize other lipid acid, for example EPA or DHA as parent material.

[0003.0.5.5] lipid acid and triacylglycerol have multiple application at food and fodder industry, cosmetic industry and medicine department.Depend on that they are saturated or unsaturated fatty acids of free or the triacylglycerol with saturated or unsaturated fatty acid content of raising, they are applicable to multiple application; Therefore, for example, polyunsaturated fatty acid (=PUFA) can add in the infant food to improve nutritive value.Multiple lipid acid and triacylglycerol mainly be obtained from microorganism such as fungi or oil-produced vegetable, comprise plant plankton and algae, for example soybean, rape, Sunflower Receptacle and other, the form of the normally triacylglycerol that obtains therein.

[0004.0.5.5] microorganism is as genus mortierella or oil-produced vegetable, if as soybean, Semen Brassicae campestris or Sunflower Receptacle or algae such as Crytocodinium belongs to or the brown algae owner contains the source of the oils of PUFA, the form of the normally triacylglycerol that obtains therein.Alternatively, can advantageously from animal such as fish, obtain them.Can advantageously prepare free fatty acids by using highly basic such as potassium hydroxide or sodium hydroxide to be hydrolyzed.Oil-produced vegetable, for example soybean, Semen Brassicae campestris, safflower or Sunflower Receptacle do not produce high polyunsaturated fatty acid, as DHA, EPA, ARA, two height-gamma-linoleic acid (C _20:3 ^{Δ 8,11,14}) or clupanodonic acid (=DPA, C _22:5 ^{Δ 7,10,13,16,19}).The natural origin of described lipid acid is fish, for example catfish, salmon, sardines, snapper, common eel, carp, trout, halibut, mackerel, pike-perch or tuna or algae.

Whether the oils that [0005.0.5.5] has unsaturated fatty acids or have a saturated fatty acid is preferred, and this depends on and is intended to purpose; Therefore, for example, the lipid that has unsaturated fatty acids, particularly polyunsaturated fatty acid in human nutrition is preferred, thereby has positive influence because they have positive influence to the cholesterol levels in the blood to cardiopathic possibility takes place.They are used for multiple nutrients food or medicine.In addition, PUFA is generally used for food, feed and cosmetic industry.Many unsaturated omega-3-fatty acids and/or ω-6-lipid acid is the integral part of animal-feed and human foods.Owing to the how unsaturated omega-3-fatty acid of human food prods's common component (they are main components of fish oil) should be added food to improve the nutritive value of food, therefore, for example polyunsaturated fatty acid such as DHA or EPA are added into as mentioned above in the formulated infant milk to improve its nutritive value.Real essential fatty acids linoleic and linolenic acid lipid acid have many active effects in human body, for example heart, artery and the skin for health has active effect.For example they can alleviate eczema, diabetic neuropathy or PMS and periodicity mastalgia.

[0006.0.5.5] polyunsaturated omega-3-fatty acid and ω-6-lipid acid is the precursor that for example is known as the paracrine hormone family of eicosanoid (for example prostaglandin(PG)), and prostaglandin(PG) is the meta-bolites of two height-gamma-linoleic acid, ARA or EPA.Eicosanoid participates in that fat splitting is regulated, inflammatory reaction is initial, the adjusting of blood circulation and blood pressure and other center function of health.Eicosanoid comprises prostaglandin(PG), leukotriene, thromboxane and prostacyclin.As if omega-3-fatty acid mainly come the atherosis and cardiovascular disorder of prevention of arterial by the level of regulating the inhomogeneity arachic acid.Other eicosanoid is thromboxane and leukotriene, and it is the meta-bolites of ARA or EPA.

[0007.0.5.5] is because the positive characteristics of polyunsaturated fatty acid has spared no effort to prepare available participation lipid acid or triacylglycerol synthetic gene, in the past so that produce oils in the multiple biology of the unsaturated fatty acid content with change.

[0008.0.5.5] in the past few years used the recombinant DNA technology method by increasing each fatty acid biological synthetic gene and study its influence that lipid acid is produced and improve oil-contg in microorganism or the plant.For example, described Δ-9-desaturase in WO 91/13972, this enzyme participates in the synthetic of polyunsaturated fatty acid.In WO 93/11245 claimed Δ-15-desaturase and in WO 94/11516 claimed Δ-12-desaturase.Other desaturase has also been described, for example at EP-A-0550162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0794250, Stukey etc., J.Biol.Chem., 265,1990:20144-20149, Wada etc., Nature 347,1990:200-203 or Huang etc., Lipids, 34, among the 1999:649-659.Yet, up to now, because the multiple desaturase that exists with the membrane bound protein form all has very large difficulty on separation and sign, therefore these enzymes have only carried out some inadequate feature descriptions (McKeon etc., Method inEnzymol.71,1981:12141-12147 on biological chemistry, Wang etc., Plant Physiol.Biochem., 26,1988:777-792).Usually, characterize, by analyzing raw material and product enzymic activity is studied then by film is imported suitable biology in conjunction with desaturase.Effect of expressing about desaturase and the influence that polyunsaturated fatty acid is formed thereof can be noticed, as institute's descriptions up to now by expressing polyunsaturated fatty acid/lipid that desaturase and prolongation enzyme have only obtained lower aq.Therefore, need alternate and more effectively have the approach of higher output yield.

[0009.0.5.5] as mentioned above, indispensable fatty acid is people and many animals, for example domestic animal is necessary.As if in the disclosed research for the middle-aged people of Finland investigator (International Journal of Cancer, on September 1st, 2004), high linoleic acid is taken in and has been reduced the danger of suffering from prostate cancer and other cancer.Positive influence (Umemura etc., Stroke, 2002, the 33 volumes, 2086-2093 page or leaf) to apoplexy is disclosed in another publication.

[0010.0.5.5] therefore, the quality of improving food and animal-feed is the vital task of food with fodder industry.This is necessary, because some lipid acid that exist in plant as mentioned above are restrictive for Mammals.Particularly advantageous to the quality of food and animal-feed is as much as possible with the fatty acid profile balance in the diet, there is not active effect because omega-3-fatty acid concentration substantially exceeds prescribed concentration in the food, unless omega-3-fatty acid content and ω-6-fatty acid content balance in the food.The further raising of quality only can realize by adding other lipid acid (it is restrictive lipid acid under these conditions).Restricted lipid acid is carried out target with the form of sintetics add and must carry out extremely modestly, unbalance to avoid lipid acid.

[0011.0.5.5] therefore for guaranteeing the high-quality of food and animal-feed, is necessary to add multiple lipid acid to be fit to biological balance mode.

[0012.0.5.5] therefore still exists great demand new and more suitable gene, and described genes encoding participates in fatty acid biological synthetic protein and can produce some lipid acid and not form unnecessary byproduct in the particular industry scale.Selecting to be used for biosynthetic gene, above-mentioned two features are particularly importants.On the one hand, but still need to obtain the development of the highest intrinsic energy polyunsaturated fatty acid, on the other hand, produce byproduct in process of production as few as possible.

Above [0013.0.0.5] sees for disclosing of this paragraph [0013.0.0.0]

[0014.0.5.5] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is linolic acid or contains linoleic triglyceride level, lipid, oils or fat.Therefore, in the present invention, term " fine chemicals " is meant " linolic acid and/or contain linoleic triglyceride level, lipid, oils or fat " as used herein.In addition, term " fine chemicals " also refers to comprise linolic acid and/or contains linoleic triglyceride level, lipid, oils or fatty fine chemicals as used herein.

[0015.0.5.5] an embodiment, term " fine chemicals " or " each fine chemicals " meaning are meant linolic acid and/or contain linoleic triglyceride level, lipid, oils or fat.In whole specification sheets, term " fine chemicals " or " each fine chemicals " meaning is meant linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, linolic acid or its salt, ester, thioesters or free form or in conjunction with the linolic acid of other compound such as triacylglycerol, glycolipid, phosphatide etc.In preferred embodiments, term " fine chemicals " meaning is meant free linoleic acid or its salt or is bonded to the linolic acid of triacylglycerol.Triacylglycerol, lipid, oils, fat or its lipid mixtures should refer to any triacylglycerol, lipid, oils and/or contain the fat of any combination or free linoleic acid, for example sphingolipid, phosphoglyceride, lipid, glycolipid class such as sphingoglycolipid, phosphatide such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, glycerine Serine, phosphatidylinositols or two glycerine Serines or monoacylglycerol, diacylglycerol or triacylglycerol or other fatty acid ester such as acetyl-CoA thioesters, it contains other saturated or unsaturated fatty acids in fatty acid molecule.

In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning be meant to have the active at least a chemical compound of above-mentioned fine chemicals.

[0016.0.5.5] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce protein b0730, b3256, YBR089C-A, YDR447C, YOR024W, b0050, b0251, b0255, b0577, b0849, b1097, b1693, b2710, b2822, b3064, b3166, b3457, b3644 and/or b4129 or have by Table I the 5th or 7 row, 63-67 and 436-449 capable shown in the activity of proteins of the coded peptide sequence of nucleic acid molecule; With

(b) producing fine chemicals in allowing described biology is to make biological growth under linolic acid or the condition that comprises linoleic fine chemicals.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II the 3rd row, 63-67 and 436-449 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, 63-67 and 436-449 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) allowing the generation fine chemicals, particularly making biological growth under the linoleic condition.

[0017.0.0.5] and [0018.0.0.5] sees top paragraph [0017.0.0.0] and [0018.0.0.0] for disclosing of [0017.0.0.5] and [0018.0.0.5] these paragraphs

The method that [0019.0.5.5] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare the fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 63-67 and 436-449 are capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 63-67 and 436-449 were capable.

[0020.0.5.5] be surprisingly found out that, and be at least a as the yeast saccharomyces cerevisiae protein shown in Table II the 3rd row, 65-67 are capable and/or at least aly give institute's conversion plant linolic acid (or fine chemicals) content raising as the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 3rd row, 63-64 and 436-449 are capable.

Above [0021.0.0.5] sees for disclosing of this paragraph [0021.0.0.0]

The sequence of [0022.0.5.5] e. coli k12 b0730 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be succinic thiokinase operon transcriptional.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary succinic thiokinase operon transcriptional or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of succinic thiokinase operon transcriptional is enhanced or produces, and for example the activity from colibacillary succinic thiokinase operon transcriptional or its homologue is enhanced or produces.

The sequence of e. coli k12 b3256 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be acetyl-CoA carboxylase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary acetyl-CoA carboxylase or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of acetyl-CoA carboxylase is enhanced or produces, and for example the activity from colibacillary acetyl-CoA carboxylase or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YBR089C-A has been published in Feldmann etc., EMBO J., and 13 (24), 5795-5809 (1994) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.It shows with Mammals high mobility group protein 1 and 2 to have homology.Its function may be unnecessary to height homologous gene NHP6A.In addition, it shows and the nonhistones chromatin protein N hp6bp homology of high speed swimming family.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YBR089C-A from yeast saccharomyces cerevisiae as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the YBR089C-A activity of proteins is enhanced or produces, and for example is enhanced or produces from the YBR089C-A protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YDR447C has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be small subunit ribosome (40s) protein 51 (rp51); Has similarity much at one and with rat S17 ribosomal protein with Rps17Ap; Rps17bp.Therefore, in one embodiment, the inventive method comprises the purposes from " ribosomal protein 51 " or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of ribosomal protein 51 is enhanced or produces, and for example is enhanced or produces from the ribosomal protein 51 of yeast saccharomyces cerevisiae or the activity of its homologue.

Yeast saccharomyces cerevisiae YOR024W sequence is committed to the EMBL protein sequence database in July, 1996 by de Haan, and its cytoactive is not still characterized.It may be a kind of membranin.Therefore, in one embodiment, the inventive method comprises the purposes of YOR024W or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of YOR024W is enhanced or produces, and for example is enhanced or produces from the YOR024W of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0050 (accession number NP_414592) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the conservative protein matter of potential participation protein-protein interaction.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary described protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, described activity of proteins is enhanced or produces, and for example the activity from colibacillary described protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b0251 (accession number NP_414785) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition HTH-type transcriptional.Therefore, in one embodiment, the inventive method comprises as shown here from the HTH-type transcription regulatory protein of colibacillary supposition or the purposes of its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of the HTH-type transcription regulatory protein of supposing is enhanced or produces, and for example is enhanced or produces from the HTH-type transcription regulatory protein of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b0255 (accession number NP_414789) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be CP4-6 prophage (prophage); The IS911 homologue.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary described CP4-6 prophage; The purposes of IS911 homologue protein or its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, described CP4-6 prophage; IS911 homologue activity of proteins is enhanced or produces, for example from colibacillary described CP4-6 prophage; The activity of IS911 homologue protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b0577 (accession number NP_415109) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition translocator.Therefore, in one embodiment, the inventive method comprises the purposes of translocator or its homologue from colibacillary supposition as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of the translocator of supposing is enhanced or produces, and for example is enhanced or produces from the translocator of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b0849 (accession number NP_415370) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase.Therefore, in one embodiment, the inventive method comprises as shown here from the glutaredoxin 1 redox coenzyme of colibacillary gsh dependency ribonucleotide reductase or the purposes of its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase is enhanced or produces, and for example is enhanced or produces from the glutaredoxin 1 redox coenzyme of colibacillary gsh dependency ribonucleotide reductase or the activity of its homologue.

The sequence of e. coli k12 b1097 (accession number NP_415615) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition thymidylate kinase.Therefore, in one embodiment, the inventive method comprises as shown here from the thymidylate kinase protein of colibacillary supposition or the purposes of its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the thymidylate kinase activity of proteins of supposing is enhanced or produces, and for example is enhanced or produces from the thymidylate kinase activity of proteins of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b1693 (accession number NP_416208) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the 3-dehydroquinate dehydratase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 3-dehydroquinate dehydratase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, 3-dehydroquinate dehydratase activity of proteins is enhanced or produces, and for example the activity from colibacillary 3-dehydroquinate dehydratase protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b2710 (accession number NP_417190) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be flavine rubredoxin (FIRd) bi-functional NO and O ₂Reductase enzyme.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary flavine rubredoxin (FIRd) bi-functional NO and O ₂The purposes of reductase enzyme protein matter or its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, flavine rubredoxin (FIRd) bi-functional NO and O ₂The activity of reductase enzyme protein matter is enhanced or produces, for example from colibacillary flavine rubredoxin (FIRd) bi-functional NO and O ₂The activity of reductase enzyme protein matter or its homologue is enhanced or produces.

The sequence of e. coli k12 b2822 (accession number NP_417299) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and it is dna helicase that its activity is defined as, ATP dependency dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary dna helicase, ATP dependency dsDNA/ssDNA exonuclease V subunit, the purposes of ssDNA endonuclease zymoprotein or its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, dna helicase, ATP dependency dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease activity of proteins is enhanced or produces, for example from colibacillary dna helicase, ATP dependency dsDNA/ssDNA exonuclease V subunit, the activity of ssDNA endonuclease zymoprotein or its homologue is enhanced or produces.

The sequence of e. coli k12 b3064 (accession number NP_417536) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the O-sialoglycoprotein endopeptidase with Actin muscle sample ATP enzymatic structure territory.Therefore, in one embodiment, the inventive method comprises as shown here from the O-sialoglycoprotein endopeptidase protein with Actin muscle sample ATP enzymatic structure territory of colibacillary supposition or the purposes of its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the O-sialoglycoprotein endopeptidase activity of proteins of supposing with Actin muscle sample ATP enzymatic structure territory is enhanced or produces, and for example is enhanced or produces from the O-sialoglycoprotein endopeptidase protein with Actin muscle sample ATP enzymatic structure territory of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b3166 (accession number NP_417635) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be tRNA Pseudouridine 5S synthase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary tRNA Pseudouridine 5S synthase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of tRNA Pseudouridine 5S synthase protein is enhanced or produces, and for example the activity from colibacillary tRNA Pseudouridine 5S synthase protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b3457 (accession number NP_417914) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary high-affinity branched-chain amino acid translocator or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of high-affinity branched-chain amino acid translocator is enhanced or produces, and for example the activity from colibacillary high-affinity branched-chain amino acid translocator or its homologue is enhanced or produces.

The sequence of e. coli k12 b3644 (accession number NP_418101) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the protein that does not characterize stress-induced.Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary stress-induced protein that does not characterize or the purposes of its homologue, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the stress-induced activity of proteins of Biao Zhenging is not enhanced or produces, and for example is enhanced or produces from the colibacillary stress-induced protein that does not characterize or the activity of its homologue.

The sequence of e. coli k12 b4129 (accession number NP_418553) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Methionin tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary Methionin tRNA synthetase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part and is linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat as described, particularly increases linolic acid and/or contains linoleic triglyceride level, lipid, oils or fat, the preferred linoleic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of Methionin tRNA synthetase protein is enhanced or produces, and for example the activity from colibacillary Methionin tRNA synthetase protein or its homologue is enhanced or produces.

The homologous compound (=homologue) of [0023.0.5.5] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.

Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with protein shown at least a Table II the 3rd row, 63-67 and 436-449 are capable, for example have by contain Table I the 5th or 7 row, 63-67 and 436-449 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in Table II the 5th or 7 row, 65-67 are capable in the polypeptide homologue of any one be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in the biology, and described homologue is from eukaryote.In one embodiment, Table II the 3rd row, the 63rd, 64 and 436-449 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 65-67 are capable is to have a same or similar active homologue, and particularly active increasing gives that fine chemicals content increases in biology or its part, and described homologue is from fungi.In one embodiment, Table II the 3rd row, the 63rd, 64 and 436-449 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Proteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 65-67 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Ascomycota.In one embodiment, Table II the 3rd row, the 63rd, 64 and 436-449 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 65-67 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from yeast.In one embodiment, Table II the 3rd row, the 63rd, 64 and 436-449 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the enterobacteria order.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 65-67 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.In one embodiment, Table II the 3rd row, the 63rd, 64 and 436-449 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from enterobacteriaceae.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 65-67 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetes.In one embodiment, Table II the 3rd row, the 63rd, 64 and 436-449 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Escherichia.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 65-67 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetaceae.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 65-67 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.

Above [0023.1.0.5] and [0024.0.0.5] sees for disclosing of [0023.1.0.5] and [0024.0.0.5] these paragraphs [0023.1.0.0] and [0024.0.0.0]

[0025.0.5.5] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause in biology or its part, the preferred described biomass cells linolic acid and/or contain that linoleic triglyceride level, lipid, oils or fat level increase and protein has as proteinic above-mentioned activity shown in Table II the 3rd row, 63-67 and 436-449 are capable, then described protein or polypeptide have " as activity of proteins shown in Table II the 3rd row, 63-67 and 436-449 are capable ".In this manual, if this kind protein or polypeptide still have Table II the 3rd row, proteinic biology or the enzyme activity shown in 63-67 and 436-449 are capable, if promptly with Table II the 3rd row, yeast saccharomyces cerevisiae protein shown in 65-67 is capable is compared and/or is listed as with Table II the 3rd, the 63rd, 64 compare with e. coli k12 protein shown in 436-449 is capable, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.5] and [0025.2.0.5] sees top paragraph [0025.1.0.0] and [0025.2.0.0] for disclosing of [0025.1.0.5] and [0025.2.0.5] these paragraphs

Paragraph [0026.0.0.0] and [0033.0.0.0] above [0026.0.0.5] to [0033.0.0.5] sees for disclosing of [0026.0.0.5] to [0033.0.0.5] these paragraphs

[0034.0.5.5] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 63-67 and protein shown in 436-449 is capable or by the active protein expression level of the protein of Table I the 5th row, 63-67 and nucleic acid molecule encoding shown in 436-449 is capable or its homologue (as Table I the 7th row, 63-67 and homologue shown in 436-449 is capable) or active aspect different, and it is difference aspect biological chemistry or genetics reason, and therefore shows the fine chemicals quantity that increases.

Paragraph [0035.0.0.0] and [0044.0.0.0] above [0035.0.0.5] to [0044.0.0.5] sees for disclosing of [0035.0.0.5] to [0044.0.0.5] these paragraphs

[0045.0.5.5] in one embodiment, at e. coli k12 protein b0730 or its homologue, succinic thiokinase operon transcriptional for example, under the situation that for example be used for C-compound and carbohydrate utilization, transcribe control, the activity of protokaryon nucleoid, transcription repressor, the transcriptional (for example shown in Table II the 5th or 7 row, the 63rd row) of regulating in conjunction with DNA is enhanced, preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 13% and 34% or higher between.

At e. coli k12 protein b3256 or its homologue, for example under the activity situation about being enhanced of acetyl-CoA carboxylase (for example shown in Table II the 5th or 7 row, the 64th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 13% and 20% or higher between.

At yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, for example show with Mammals high mobility group protein 1 and 2, gene NHP6A and the non-histone chromatin protein Nhp6bp of high speed swimming family and have under the situation that the activity of " not profiling protein matter YBR089C-A " (for example shown in Table II the 5th or 7 row, the 65th row) of homology is enhanced, preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 36% and 70% or higher between.

At yeast saccharomyces cerevisiae protein YDR447C or its homologue, for example under the activity situation about being enhanced of small subunit ribosome (40s) protein 51 (rp51) (for example shown in Table II the 5th or 7 row, the 66th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 18% and 140% or higher between.

At yeast saccharomyces cerevisiae protein YOR024W or its homologue, under the activity situation about being enhanced of for example possible membranin " not profiling protein matter YOR024W " (for example shown in Table II the 5th or 7 row, the 67th row), preferably, in one embodiment, give fine chemicals, preferred linolic acid be increased in 16% and 33% or higher between.

At e. coli k12 protein b0050 or its homologue, under the activity situation about being enhanced of the conservative protein matter of for example potential participation protein-protein interaction (for example shown in Table II the 5th or 7 row, the 436th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 15% and 37% or higher between.

At e. coli k12 protein b0251 or its homologue, for example under the activity situation about being enhanced of Jia Ding HTH-type transcriptional (for example shown in Table II the 5th or 7 row, the 437th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 14% and 31% or higher between.

E. coli k12 protein b0255 or its homologue, for example CP4-6 prophage; Under the activity situation about being enhanced of IS911 homologue (for example shown in Table II the 5th or 7 row, the 438th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 22% and 31% or higher between.

At e. coli k12 protein b0577 or its homologue, for example under the activity situation about being enhanced of Jia Ding translocator (for example shown in Table II the 5th or 7 row, the 439th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 17% and 40% or higher between.

At e. coli k12 protein b0849 or its homologue, for example under the activity situation about being enhanced of the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase (for example shown in Table II the 5th or 7 row, the 440th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 19% and 34% or higher between.

At e. coli k12 protein b1097 or its homologue, for example under the activity situation about being enhanced of Jia Ding thymidylate kinase (for example shown in Table II the 5th or 7 row, the 441st row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 15% and 30% or higher between.

At e. coli k12 protein b1693 or its homologue, for example under the activity situation about being enhanced of 3-dehydroquinate dehydratase (for example shown in Table II the 5th or 7 row, the 442nd row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 17% and 32% or higher between.

At e. coli k12 protein b2710 or its homologue, for example flavine rubredoxin (FIRd) bi-functional NO and O ₂Under the activity situation about being enhanced of reductase enzyme (for example shown in Table II the 5th or 7 row, the 443rd row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 15% and 29% or higher between.

At e. coli k12 protein b2822 or its homologue, dna helicase for example, ATP dependency dsDNA/ssDNA exonuclease V subunit, under the activity situation about being enhanced of ssDNA endonuclease (for example shown in Table II the 5th or 7 row, the 444th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 15% and 27% or higher between.

At e. coli k12 protein b3064 or its homologue, for example under the activity situation about being enhanced of Jia Ding O-sialoglycoprotein endopeptidase (for example shown in Table II the 5th or 7 row, the 445th row) with Actin muscle sample ATP enzymatic structure territory, preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 17% and 30% or higher between.

At e. coli k12 protein b3166 or its homologue, for example under the activity situation about being enhanced of tRNA Pseudouridine 5S synthase (for example shown in Table II the 5th or 7 row, the 446th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 14% and 21% or higher between.

At e. coli k12 protein b3457 or its homologue, for example under the activity situation about being enhanced of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) (for example shown in Table II the 5th or 7 row, the 447th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 20% and 44% or higher between.

At e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 448th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 14% and 35% or higher between.

At e. coli k12 protein b4129 or its homologue, for example under the activity situation about being enhanced of Methionin tRNA synthetic enzyme (for example shown in Table II the 5th or 7 row, the 449th row), preferably, give in one embodiment fine chemicals, preferred linolic acid be increased in 20% and 47% or higher between.

[0046.0.5.5] in one embodiment, at e. coli k12 protein b0730 or its homologue, for example under the situation that the activity of succinic thiokinase operon transcriptional is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b3256 or its homologue, for example under the situation that the activity of acetyl-CoA carboxylase is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, for example under the situation that the activity of " not profiling protein matter YBR089C-A " is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR447C or its homologue, for example under the situation that the activity of small subunit ribosome (40s) protein 51 (rp51) is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR024W or its homologue, for example under the situation that the activity of " not profiling protein matter YOR024W " is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b0050 or its homologue, under the situation that the activity of the conservative protein matter of for example potential participation protein-protein interaction is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b0251 or its homologue, for example under the situation that the activity of Jia Ding HTH-type transcriptional is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, e. coli k12 protein b0255 or its homologue, for example CP4-6 prophage; Under the situation that the activity of IS911 homologue is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b0577 or its homologue, for example under the situation that the activity of Jia Ding translocator is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b0849 or its homologue, for example under the situation that the activity of the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b1097 or its homologue, for example under the situation that the activity of Jia Ding thymidylate kinase is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b1693 or its homologue, for example under the situation that the activity of 3-dehydroquinate dehydratase is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b2710 or its homologue, for example flavine rubredoxin (FIRd) bi-functional NO and O ₂Under the situation that the activity of reductase enzyme is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b2822 or its homologue, dna helicase for example, ATP dependency dsDNA/ssDNA exonuclease V subunit, under the situation that the activity of ssDNA endonuclease is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b3064 or its homologue, for example under the situation that the activity of Jia Ding the O-sialoglycoprotein endopeptidase with Actin muscle sample ATP enzymatic structure territory is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b3166 or its homologue, for example under the situation that the activity of tRNA Pseudouridine 5S synthase is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b3457 or its homologue, for example under the situation that the activity of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b3644 or its homologue, for example under the situation that the stress-induced activity of proteins that does not characterize is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

In one embodiment, at e. coli k12 protein b4129 or its homologue, for example under the situation that the activity of Methionin tRNA synthetic enzyme is enhanced, preferably give fine chemicals and contain linoleic triglyceride level, lipid, oils and/or fat increase.

[0047.0.0.5] and [0048.0.0.5] sees top paragraph [0047.0.0.0] and [0048.0.0.0] for disclosing of [0047.0.0.5] and [0048.0.0.5] these paragraphs

[0049.0.5.5] has to give and improves each fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, the polypeptide of consensus sequence shown in 63-67 and 436-449 are capable, perhaps as Table II the 5th or 7 row, its function homologue shown in 63-67 and 436-449 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 63-67 and 436-449 are capable or its function homologue as herein described) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.5.5] for the purposes of the present invention, term " linolic acid " also comprises corresponding salt, for example linoleic sylvite or sodium salt or have amine such as the linoleic salt of diethylamine.

[0051.0.5.5] and [0052.0.0.5] sees top paragraph [0051.0.0.0] and [0052.0.0.0] for disclosing of [0051.0.5.5] and [0052.0.0.5] these paragraphs

[0053.0.5.5] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having as the active polypeptide of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in)) and is expressed and increase, and has the activity of the raising fine chemicals described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example having as the active polypeptide of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in)) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of the raising linolic acid described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give the raising linolic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having) as the active polypeptide of protein or its homologue shown in Table II the 3rd row, 63-67 and 436-449 are capable (for example as Table II the 7th row, 63-67 and 436-449 are capable shown in), perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give the raising linolic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give the raising linolic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in);

(f) express the transgenosis of coded protein, described protein give the raising linolic acid that has described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have the raising linolic acid described in the literary composition active, by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide or the used polypeptide of the inventive method (for example having as the active polypeptide of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in)).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.5.5] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example after active expression of polypeptides of protein shown in Table II the 3rd row, 63-67 and 436-449 are capable or its homologue (for example Table II the 5th or 7 row, 63-67 and 436-449 capable shown in polypeptide) or activity, give linolic acid and increase improving coded protein or have.

Paragraph [0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.5] to [0067.0.0.5] saw for disclosing of [0055.0.0.5] to [0067.0.0.5] these paragraphs

[0068.0.5.5] and [0069.0.5.5] sees top paragraph [0068.0.0.0] and [0069.0.0.0] for disclosing of [0068.0.5.5] and [0069.0.5.5] these paragraphs

[0070.0.5.5] imports biology separately or with other assortment of genes owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention as described below or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used expression of polypeptides of the inventive method, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example contain the lipid acid such as the palmitinic acid of high level (from the physiology of nutrition angle), Zoomeric acid, stearic acid and/or oleic favourable fatty acid composition.

Above [0071.0.5.5] sees for disclosing of this paragraph [0071.0.0.0]

[0072.0.5.5] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds except linolic acid, contain linoleic triacylglycerol, lipid, oils and/or the fat and to also have other compound, for example palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid.

[0073.0.5.5] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of protein with polypeptide of the present invention or the used polypeptide active of the inventive method or its homologue (for example Table II the 5th or 7 row, 63-67 and 436-449 capable shown in) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of promptly giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and other optional free and/or conjugated fatty acid.

[0074.0.5.5] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or bonded fine chemicals or free and bonded fine chemicals, if and dissociate or/and conjugated fatty acid, particularly oleic acid as selecting to produce, reclaim and expect to separate other.

Paragraph [0075.0.0.0] was to [0084.0.0.0] above [0075.0.0.5] to [0084.0.0.5] saw for disclosing of [0075.0.0.5] to [0084.0.0.5] these paragraphs

[0085.0.5.5] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, 63-67 and 436-449 line description and shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, 63-67 and 436-449 line description with shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps (c) (a) with (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.5] and [0087.0.0.5] sees top paragraph [0086.0.0.0] and [0087.0.0.0] for disclosing of [0086.0.0.5] and [0087.0.0.5] these paragraphs

[0088.0.5.5] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified fatty acid content.Because the nutritive value that for example is used for the plant of poultry depends on above-mentioned indispensable fatty acid and as the big scale of construction of the lipid acid in fodder energy source, so this is very important for the plant breeder.Improve or produced as Table II the 5th or 7 row, 63-67 and 436-449 capable shown in after the activity of polypeptide, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, thus the transgenic plant that produced grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0088.1.0.5], [0089.0.0.5], [0090.0.0.5] and [0091.0.5.5] be for [0088.1.0.5],

[0089.0.0.5], top paragraph is seen in disclosing of [0090.0.0.5] and [0091.0.5.5] these paragraphs

[0088.1.0.0], [0089.0.0.0], [0090.0.0.0] and [0091.0.0.0]

Paragraph [0092.0.0.0] was to [0094.0.0.0] above [0092.0.0.5] to [0094.0.0.5] saw for disclosing of [0092.0.0.5] to [0094.0.0.5] these paragraphs

[0095.0.5.5] advantageously increases the free fatty acids pond to separate a large amount of pure fine chemicals by method of the present invention in genetically modified organism.

[0096.0.5.5] is in another embodiment preferred of the present invention, improve nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide, for example lipid acid transporter albumen or compound (as desired fats acid in the biology as linolic acid or linolenic pond) are combined and be can be used for producing each fine chemicals and (consult BaO and Ohlrogge, Plant Physiol.1999 August; 120 (4): 1057-1062). this type of lipid acid transporter protein can play ligation between the so-called storehouse tissue of the synthetic place of lipid acid and depot fat acid, triacylglycerol, oils and fat.

[0097.0.5.5] sees [0097.0.0.0] for disclosing of this paragraph.

[0098.0.5.5] in preferred embodiments, fine chemicals (linolic acid) be produce according to the present invention and carry out isolating where necessary.It is favourable as palmitinic acid, stearic acid, Zoomeric acid and/or oleic acid mixture or other fatty acid mixt that the method according to this invention produces other lipid acid.

For microbial fermentation, aforementioned fatty acids can be accumulated in substratum and/or the cell [0099.0.5.5].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.The compound that can handle spissated fermented liquid and be advantageously used in preparation by freeze-drying, spraying drying, spraying choosing grain or additive method subsequently.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps separation of fatty acids or fatty acid composition from biology and substratum.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.5.5] comprises the transgenic plant of the method according to this invention synthetic lipid acid such as saturated or polyunsaturated fatty acid and can advantageously directly put on market, and do not need institute's synthetic oils, lipid or lipid acid are separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.Yet, the fine chemicals that produces according to the inventive method can also from biological, advantageously from plant with the isolated in form of oils, fat, lipid and/or free fatty acids.Yet the fine chemicals that the method according to this invention produces can also be with the form of the form of its oils, fat, lipid and/or free fatty acids from biology, advantageously separate from plant.Can obtain the lipid acid that produces by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part, preferred plant seed.In order to improve the efficient that oil extracts, with vegetable material, particularly seed clean, softening (temper) and to shell in case of necessity and peel off be favourable.In this article, oils, fat, lipid and/or free fatty acids can need not heat cold be beaten or cold-press process obtains by being called.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.In this mode, can separate more than 96% of compound that the inventive method produces.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.Afterwards, randomly, free fatty acids can be by advantageously using down alkali (as water-soluble KOH or NaOH) and acid hydrolysis processing or remove by enzymic hydrolysis at alcohol (for example methyl alcohol and ethanol), and by for example being separated and subsequently acidifying (is for example passed through H ₂SO ₄) separate.Can also not use above-mentioned procedure of processing and release fat acid.If expectation can water thoroughly be washed products therefrom to remove residual soap and alkali in the product, and is dry then.Be to remove residual pigment in the product, can use Fuller's earth (filler ' s earth) or activated carbon bleaching.At last under vacuum vapor distillation with the product deodorizing.The lipid acid of these chemical purifications or fatty acid composition can advantageously be applied to grocery trade, cosmetic industry, particularly pharmacy industry.

[0101.0.5.5] sees [0101.0.0.0] for disclosing of this paragraph.

[0102.0.5.5] for example, lipid acid can advantageously detect by the GC separation method.By using standard method of analysis GC, GC-MS or TLC can clearly detect the existence of fatty acids products to the biology analysis of recombinating, described analytical procedure Christie and reference wherein are described (1997, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric method], Lipide33:343-353).An embodiment is by FAME and GC-MS or TLC (abbreviation: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography) analysis lipid acid.By ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material fragmentation to be analyzed.After the fragmentation, must material is centrifugal.With pellet resuspended in distilled water, in 100 ℃ of heating 10 minutes, cooled on ice was also centrifugal, then extracts 1 hour in 90 ℃ in containing the 0.5M sulfuric acid (in methyl alcohol) of 2% Propanal dimethyl acetal, this causes producing the oil and the fat compound of hydrolysis, and this can obtain transmethylase fat.In sherwood oil, extract these fatty acid methyl esters and finally use capillary column (Chrompack, the WCOT fused silica, CP-Wax-52CB, 25 μ m, 0.32mm) gradient temperature between 170 ℃ and 240 ℃ 20 minutes and 240 ℃ carried out the GC analysis in following 5 minutes.The identity of resulting fatty acid methyl ester must be used and can define from the standard that commercial source (being Sigma) obtains.

[0103.0.5.5] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as the sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as the sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer as sequence shown in Table III the 7th row, 63-67 and 436-449 are capable obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as the consensus sequence of sequence shown in Table IV the 7th row, 63-67 and 436-449 are capable and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, 63-67 and 436-449 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0103.1.0.5] and [0103.2.0.5] sees top paragraph [0103.1.0.0] and [0103.2.0.0] for disclosing of [0103.1.0.5] and [0103.2.0.5] these paragraphs

[0104.0.5.5] in one embodiment, sequence was different shown in the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 63-67 and 436-449 were capable, and is preferably different with sequence shown in Table I A the 5th or 7 row, 63-67 and 436-449 are capable.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 63-67 and 436-449 capable shown in sequence, preferred Table I A the 5th or 7 row, 63-67 and 436-449 capable shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 63-67 and 436-449 capable shown in sequence, preferred Table I A the 5th or 7 row, 63-67 and 436-449 capable shown in polypeptide of sequence.

Paragraph [0105.0.0.0] and [0107.0.0.0] above [0105.0.0.5] to [0107.0.0.5] sees for disclosing of [0105.0.0.5] to [0107.0.0.5] these paragraphs

Advantageously improved in [0108.0.5.5] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in 63-67 and 436-449 are capable, from Table II the 5th or 7 row, aminoacid sequence shown in 63-67 and 436-449 are capable is derived or is listed as from containing Table IV the 7th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 63-67 and 436-449 are capable, perhaps its coding has as Table II the 5th or 7 row, protein enzymic activity shown in 63-67 and 436-449 are capable or bioactive polypeptide or for example give derivative or the homologue that the fine chemicals linolic acid increases behind its expression or active the increasing.

[0109.0.5.5] meets personally [0109.0.0.0] for disclosing of this paragraph

[0110.0.5.5] helps the nucleic acid molecule of the polypeptide that the inventive method and coding have the used polypeptide active of the used or of the present invention process of the inventive method (for example as protein shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or by as the protein of nucleic acid molecule encoding shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable or its homologue (shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable)) and can determine from generally open database.

Above [0111.0.0.5] sees for disclosing of this paragraph [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.5.5] the inventive method is the isolated nucleic acid sequences form, its coding has as the polypeptide of polypeptide active shown in Table II the 3rd row, 63-67 and 436-449 are capable or has polypeptide as peptide sequence shown in Table II the 5th and 7 row, 63-67 and 436-449 are capable, and gives each fine chemicals and increase.

Paragraph [0113.0.0.0] and [0120.0.0.0] above [0113.0.0.5] to [0120.0.0.5] sees for disclosing of [0113.0.0.5] to [0120.0.0.5] these paragraphs

[0121.0.5.5] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with the difference that one or more amino acid moleculars are arranged as peptide sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or its functional homologue as herein described, described artificial sequence is preferably given aforementioned activity, and promptly giving each fine chemicals after improving its activity increases.

Paragraph [0122.0.0.0] and [0127.0.0.0] above [0122.0.0.5] to [0127.0.0.5] sees for disclosing of [0122.0.0.5] to [0127.0.0.5] these paragraphs

The synthetic oligonucleotide primer thing that [0128.0.5.5] polymerase chain reaction (PCR) amplification is used (for example as the primer shown in Table III the 7th row, 63-67 and 436-449 are capable) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 63-67 and 436-449 capable shown in sequence or produce as sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable.

[0129.0.5.5] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (sequence of polypeptide particularly of the present invention or the used polypeptide of the inventive method).Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence shown in Table IV the 7th row, 63-67 and 436-449 are capable is from described comparison and represent this type of conservative region.

[0130.0.5.5] sees [0130.0.0.0] for disclosing of this paragraph.

Paragraph [0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.5] to [0138.0.0.5] saw for disclosing of [0131.0.0.5] to [0138.0.0.5] these paragraphs

[0139.0.5.5] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 5th or 7 row, 63-67 and 436-449 capable shown in sequence hybridization, and coding is expressed and is had the active peptide of the linolic acid of increasing.

[0140.0.0.5] to [0146.0.0.5] is for disclose above the section of opinion [0140.0.0.0] and [0146.0.0.0] of [0140.0.0.5] to [0146.0.0.5] these paragraphs

[0147.0.5.5] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 5th or 7 row, 63-67 and 436-449 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.5.5] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 63-67 and 436-449 are capable, preferred Table I B the 5th or 7 row, nucleotide sequence shown in 63-67 and 436-449 are capable or its funtion part homology are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases fine chemicals.

[0149.0.5.5] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 5th or 7 row, 63-67 and 436-449 capable shown in one of nucleotide sequence or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (increasing as giving each fine chemicals) and as Table II the 5th or 7 row, 63-67 with 436-449 is capable, preferred Table II B the 5th or 7 row, 63-67 and the 436-449 protein shown in capable.

[00149.1.0.5] randomly, with Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 5th or 7 row, 63-67 and 436-449 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity is for as Table II the 3rd row, 63-67 with 436-449 is capable, preferred Table II B the 3rd row, 63-67 and the 436-449 protein shown in capable is known activity or is used for these protein of note.

[0150.0.5.5] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 5th or 7 row, 63-67 and 436-449 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving linolic acid when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 63-67 and 436-449 capable shown in sense strand, Table I the 5th or 7 row, 63-67 and the 436-449 of one of sequence capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 63-67 and 436-449 capable shown in the right PCR of primer will produce as Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 7th is listed as, 63-67 and 436-449 capable shown in the fragment of polynucleotide sequence.

[0151.0.0.5] meets personally [0151.0.0.0] for disclosing of this paragraph

[0152.0.5.5] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as the abundant homology of aminoacid sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the linoleic activity of raising as be shown in the examples.

[0153.0.5.5] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as protein shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or its part have Table II the 5th for example as herein described or 7 row, 63-67 and 436-449 capable shown in the activity of polypeptide.

[0154.0.5.5] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.5] and [0156.0.0.5] sees top paragraph [0155.0.0.0] and [0156.0.0.0] for disclosing of [0155.0.0.5] and [0156.0.0.5] these paragraphs

[0157.0.5.5] the present invention relate in addition owing to the genetic code degeneracy be different from Table I the 5th or 7 row, 63-67 and 436-449 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase) for example comprises as the polypeptide of consensus sequence shown in Table IV the 7th row, 63-67 and 436-449 are capable or as the nucleic acid molecule of polypeptide shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as the consensus sequence shown in Table IV the 7th row, 63-67 and 436-449 are capable or as polypeptide or its function homologue shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as the consensus sequence shown in Table IV the 5th or 7 row, 63-67 and 436-449 are capable or as the basic homology of aminoacid sequence of polypeptide shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I A the 5th or 7 row, 63-67 and the 436-449 sequence shown in capable.Preferably, nucleic acid molecule of the present invention is a functional homologous compound or identical with nucleic acid molecule shown in Table I B the 5th or 7 row, 63-67 and 436-449 are capable.

Paragraph [0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.5] to [0160.0.0.5] saw for disclosing of [0158.0.0.5] to [0160.0.0.5] these paragraphs

[0161.0.5.5] therefore, in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor length are at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 63-67 and 436-449 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

Above [0162.0.0.5] sees for disclosing of this paragraph [0162.0.0.0]

[0163.0.5.5] preferably, under stringent condition with Table I the 5th or 7 row, 63-67 and 436-449 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

Above [0164.0.0.5] sees for disclosing of this paragraph [0164.0.0.0]

[0165.0.5.5] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 63-67 and 436-449 capable shown in sequence).

[0166.0.0.5] and [0167.0.0.5] sees top paragraph [0166.0.0.0] and [0167.0.0.0] for disclosing of [0166.0.0.5] and [0167.0.0.5] these paragraphs

[0168.0.5.5] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are with different as the contained sequence of sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable, but have kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the aminoacid sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding is with identical at least about 60% as the sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable, more preferably with identical at least about 70% as one of sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable, even more preferably with as the sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable at least about 80%, 90% or 95% homology, and most preferably with identical at least about 96%, 97%, 98% or 99% as the sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table II B the 7th row, 63-67 and 436-449 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 63-67 with 436-449 is capable, preferred Table II B the 7th row, 63-67 and the 436-449 aminoacid sequence shown in capable is at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 63-67 and 436-449 are capable, preferred Table II B the 7th row, 63-67 is identical at least about 60% with the sequence shown in 436-449 is capable, more preferably with as Table II the 5th or 7 be listed as, 63-67 and 436-449 are capable, preferred Table II B the 7th row, 63-67 is identical at least about 70% with one of sequence shown in 436-449 is capable, even more preferably with as Table II the 5th or 7 be listed as, 63-67 and 436-449 are capable, preferred Table II B the 7th row, sequence shown in 63-67 and 436-449 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 63-67 and 436-449 are capable, preferred Table II B the 7th row, sequence shown in 63-67 and 436-449 are capable is at least about 96%, 97%, 98% or 99% is identical.

Paragraph [0169.0.0.0] was to [0175.0.0.0] above [0169.0.0.5] to [0175.0.5.5] saw for disclosing of [0169.0.0.5] to [0175.0.5.5] these paragraphs

[0176.0.5.5] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in 63-67 and 436-449 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 63-67 and 436-449 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in 63-67 and 436-449 are capable has essentially identical character and discerns.

[0177.0.5.5] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the functional equivalent that nucleotide sequence obtained shown in 63-67 and 436-449 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 63-67 and 436-449 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in 63-67 and 436-449 are capable.

Above [0178.0.0.5] sees for disclosing of this paragraph [0178.0.0.0]

[0179.0.5.5] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I the 5th or 7 row, 63-67 and 436-449 capable shown in), and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table II B the 7th is listed as, 63-67 and 436-449 capable shown in the nucleic acid molecule of homologous protein of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to encoding sequence, in sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable, introduce sudden change.

Paragraph [0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.5] to [0183.0.0.5] saw for disclosing of [0180.0.0.5] to [0183.0.0.5] these paragraphs

[0184.0.5.5] is employed to have as Table I the 5th or 7 row, 63-67 and 436-449 are capable, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in 63-67 and 436-449 are capable, perhaps come Table II the 5th or 7 row freely, 63-67 and 436-449 are capable, preferred Table II B the 7th row, the homologous compound of the nucleotide sequence of sequence shown in 63-67 and 436-449 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.5.5] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I the 5th or 7 row, 63-67 with 436-449 is capable, preferred Table I B the 7th row, 63-67 and the 436-449 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 63-67 with 436-449 is capable, other nucleotide sequence of not showing in preferred Table I B the 7th row, 63-67 and the 436-449 arbitrary sequence shown in capable.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 63-67 with 436-449 is capable, preferred Table I B the 7th row, 63-67 and the 436-449 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.5.5] also preferred the inventive method comprise as Table II the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table II B the 7th row, 63-67 and 436-449 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table II B the 7th row, 63-67 and 436-449 capable shown in sequence identical.

[0187.0.5.5] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table II B the 7th row, 63-67 and 436-449 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table II B the 7th row, 63-67 and 436-449 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.5.5] still has the basic biologic activity of polypeptide of the present invention (being its active basic reduction that do not have) of giving fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active comparing with polypeptide expressed activity as Table II the 5th or 7 row, 63-67 and shown in 436-449 is capable and under the same conditions do not reduced substantially.In one embodiment, polypeptide of the present invention is to comprise as sequence shown in Table II B the 7th row, 63-67 and 436-449 are capable or by its homologue of forming.

[0189.0.5.5] as the homologous compound of sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.5], [0191.0.5.5], [00191.1.0.5] and [0192.0.0.5] to [0203.0.0.5] is for [0190.0.0.5], [0191.0.5.5], [0191.1.0.5] and [0192.0.0.5] be paragraph [0190.0.0.0] above disclosing of [0203.0.0.5] these paragraphs seen, [0191.0.0.0], [0191.1.0.0] and [0192.0.0.0] is to [0203.0.0.0]

[0204.0.5.5] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table II B the 7th row, 63-67 and 436-449 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given fine chemicals quantity in biological or its part increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 7th row, 63-67 and 436-449 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as the primer shown in Table III the 7th row, 63-67 and 436-449 are capable or primer amplification from cDNA library or genomic library is obtained by using, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 63-67 and 436-449 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described peptide coding is as Table II the 5th or 7 row, 63-67 and 436-449 is capable, the structural domain of preferred Table II B the 7th row, 63-67 and the 436-449 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, nucleic acid molecule shown in 63-67 and 436-449 are capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 63-67 and 436-449 are capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt; Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A or IB the 5th or 7 row, 63-67 and 436-449 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule do not form by Table I A or IB the 5th or 7 row, 63-67 and 436-449 by the sequence shown in capable.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 63-67 and 436-449 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as the peptide sequence shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as the protein of sequence shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable.

In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable at least 30% identical and with Table II A or IIB the 5th or 7 row, 63-67 and 436-449 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

Paragraph [0205.0.0.0] was to [0226.0.0.0] above [0205.0.0.5] to [0226.0.0.5] saw for disclosing of [0205.0.0.5] to [0226.0.0.5] these paragraphs

[0227.0.5.5] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 63-67 and the capable sequence or derivatives thereof of mentioning of 436-449, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid biosynthetic pathway of at least one lipid acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic each required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 63-67 and 436-449 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.5.5] therefore cultivated spontaneous the mistake and expressed coding and fatty acid metabolism (particularly lipid acid is synthetic) at least one nucleic acid of related protein or the biology of a gene in another embodiment of the present invention.

[0229.0.5.5] can be the coding saturated fatty acid with other favourable nucleotide sequences that sequence that present method is used and/or the combination of aforementioned biosynthesis gene are expressed; the sequence of biosynthetic other gene of polyunsaturated fatty acid; for example as WO 98/46765; WO 98/46763; WO 98/46764; WO 99/64616; WO 00/20603; WO 00/20602; WO 00/40705; US20040172682; US 20020156254; US 6; 677,145; desaturase of describing among US 20040053379 or the US20030101486 such as Δ-4-desaturase; Δ-5-desaturase; Δ-6-desaturase; Δ-8-desaturase; Δ-9-desaturase; Δ-12-desaturase; Δ-17-desaturase; ω-3-desaturase; prolong enzyme such as Δ-5-and prolong enzyme; Δ-6-prolongs enzyme; Δ-9-prolongs enzyme; acyl group-CoA-desaturase; acyl-acp-desaturase; acyl-acp-thioesterase; the fatty acid acyl based transferase; acyl group-CoA lysophospholipid acyltransferase; acyl group-CoA carboxylase; Fatty acid synthetase; fatty acid hydroxylase; acyl group-CoA oxydase; acetylenase; lipoxygenase; three acyl group lipase etc.These genes cause the synthetic increase of indispensable fatty acid.

Above [0230.0.5.5] sees for disclosing of this paragraph [0230.0.0.0]

[0231.0.5.5] in another advantageous embodiment of the inventive method, the employed biology of present method is to have weakened the linoleic protein of degraded simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

Paragraph [0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.5] to [0276.0.0.5] saw for disclosing of [0232.0.0.5] to [0276.0.0.5] these paragraphs

[0277.0.5.5] can separate the lipid acid that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.The fine chemicals that produces according to the inventive method can be as previously mentioned from biological, advantageously from plant with the isolated in form of oils, fat, lipid and/or free fatty acids.The lipid acid of producing by this process can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing or extract realization with plant part, preferred plant seed.Preferred hexane wherein can separate more than 96% of compound that process of the present invention produces as the solvent in this process.Afterwards, resulting product is further processed, promptly come unstuck, make with extra care, bleaching and/or deodorizing.

Paragraph [0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.5] to [0282.0.0.5] saw for disclosing of [0278.0.0.5] to [0282.0.0.5] these paragraphs

[0283.0.5.5] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-antibody as polypeptide shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or its antigen part, polypeptide (polypeptide for example of the present invention or its fragment) generation that it can comprise above-mentioned sequence or be made up of above-mentioned sequence by the standard technique utilization.Preferably specificity is in conjunction with the monoclonal antibody as polypeptide shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable.

Above [0284.0.0.5] sees for disclosing of this paragraph [0284.0.0.0]

[0285.0.5.5] the present invention relates to have as sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable in one embodiment.

[0286.0.5.5] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in 63-67 and 436-449 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in 63-67 and 436-449 are capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to and comprising more than a polypeptide as consensus sequence shown in Table IV the 7th row, 63-67 and 436-449 are capable.

Paragraph [0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.5] to [0290.0.0.5] saw for disclosing of [0287.0.0.5] to [0290.0.0.5] these paragraphs

[0291.0.5.5] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise and contain plant or microorganism specificity consensus sequence or by its polypeptide of forming.In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in 63-67 and 436-449 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in 63-67 and 436-449 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable.

Above [0292.0.0.5] sees for disclosing of this paragraph [0292.0.0.0]

[0293.0.5.5] the present invention relates to give the polypeptide that each fine chemicals increases in one embodiment in biological or its part, described polypeptide is by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention coding.

In one embodiment, polypeptide of the present invention have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help to form as the sequence of nucleic acid molecule encoding shown in Table I A or IB the 5th or 7 row, 63-67 and 436-449 are capable.

[0294.0.5.5] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, the polypeptide of activity of proteins shown in 63-67 and 436-449 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in 63-67 and 436-449 are capable.

[0295.0.0.5], [0296.0.0.5] and [0297.0.5.5] be for [0295.0.0.5], [0296.0.0.5] and

Disclosing of [0297.0.5.5] these paragraphs sees that top paragraph [0295.0.0.0] is to [0297.0.0.0]

The chemical of [00297.1.0.5] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 63-67 and 436-449 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.

[0298.0.5.5] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II the 5th or 7 row, 63-67 and 436-449, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable.

[0299.0.5.5] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of aminoacid sequence shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable.

[0300.0.5.5] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,63-67 and 436-449 capable shown in sequence different.Therefore, this polypeptide contains with complete amino acid sequence homology as sequence shown in Table II A or IIB the 5th or 7 row, 63-67 and 436-449 are capable and is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

Above [0301.0.0.5] sees for disclosing of this paragraph [0301.0.0.0]

The biologically-active moiety of [0302.0.5.5] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence of the aminoacid sequence shown in 63-67 and 436-449 are capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

Above [0303.0.0.5] sees for disclosing of this paragraph [0303.0.0.0]

[0304.0.5.5] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as the active of polypeptide shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.5.5], [0306.0.5.5] and [0306.1.0.5] are for [0305.0.5.5], and disclosing of [0306.0.5.5] and [0306.1.0.5] these paragraphs seen top paragraph [0305.0.0.0], [0306.0.0.0] and [0306.1.0.0]

[0307.0.0.5] and [0308.0.0.5] sees top paragraph [0307.0.0.0and[0308.0.0.0] for disclosing of [0307.0.0.5] and [0308.0.0.5] these paragraphs

[0309.0.0.5] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned, as Table II the 5th or 7 row, be meant the polypeptide that has corresponding polypeptide of the present invention or be used for the amino acid sequence of polypeptide of the inventive method shown in 63-67 and 436-449 are capable, be not listed as and be shown in Table II the 5th or 7, " non-polypeptide " during 63-67 and 436-449 are capable or " other polypeptide " are meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with the homology not basically of the polypeptide with protein active, for example do not give described in the literary composition active and from the protein of identical or different biology.

Paragraph [0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.5] to [0334.0.0.5] saw for disclosing of [0310.0.0.5] to [0334.0.0.5] these paragraphs

[0335.0.5.5] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of one of sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

Paragraph [0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.5] to [0342.0.0.5] saw for disclosing of [0336.0.0.5] to [0342.0.0.5] these paragraphs

[0343.0.5.5] as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 63-67 and 436-449 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as one of sequence shown in Table I the 5th or 7 row, 63-67 and 436-449 are capable or its homologue in a kind of organism.

[0344.0.0.5] is to [0350.0.0.5], [0351.0.5.5] and [0352.0.0.5] to [0361.0.0.5] for [0344.0.0.5] to [0350.0.0.5], [0351.0.5.5] and [0352.0.0.5] above disclosing of [0361.0.0.5] these paragraphs seen paragraph [0344.0.0.0] to [0361.0.0.0]

[0362.0.5.5] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or each fine chemicals of its part increases) carry out genetically modified any cell, the nucleic acid molecule of described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide (for example coding has the polypeptide of protein as polypeptide active shown in Table II the 3rd row, 63-67 and 436-449 are capable).Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, cytoactive improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Transgenosis with the active polypeptide of protein (as polypeptide shown in Table II the 3rd row, 63-67 and 436-449 are capable) is meant in the text because genomic regulation and control or manipulation, improves having protein (as Table II the 3rd row, 63-67 and polypeptide shown in 436-449 is capable) activity active or similar active polypeptide in cell or biological or its part.Example and the inventive method are described in above.

[0363.0.0.5], [0364.0.5.5] and [0365.0.0.5] to [0379.0.5.5] for [0363.0.0.5], [0364.0.5.5] and [0365.0.0.5] above disclosing of [0379.0.5.5] these paragraphs seen paragraph [0363.0.0.0] to [0379.0.0.0]

The suitable synthetic parent material of the lipid acid that [0380.0.5.5] obtains in the method for the invention as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises fatty acid composition that separation produces or fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is lipid acid or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine that the inventive method produces.

[0381.0.0.5] and [0382.0.0.5] sees top paragraph [0381.0.0.0] and [0382.0.0.0] for disclosing of [0381.0.0.5] and [0382.0.0.5] these paragraphs

[0383.0.5.5] can use organic compound in order to prepare the fine chemicals of fatty acids compound, particularly fine chemicals, and the lipid acid that for example contains as precursor compound (for example has C ₁₀--C ₁₆The lipid acid of carbon atom main chain) and/or oils, fat and/or the lipid of little organic acid (for example acetate, propionic acid or butyric acid) as fatty acid source.

[0384.0.0.5] and [0385.0.5.5] sees top paragraph [0384.0.0.0] and [0385.0.0.0] for disclosing of [0384.0.0.5] and [0385.0.5.5] these paragraphs

[0386.0.5.5] yet, can also be further purified the lipid acid of generation.For this reason, the composition that will contain product carries out thin-layer chromatography or carries out Florisil column chromatography (Bouhours J.F., J.Chromatrogr.1979,169,462) on silica-gel plate, and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.An alternative approach of purification of fatty acid is for for example carrying out crystallization under the urea existence condition.These methods can combination with one another.

Paragraph [0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.5] to [0392.0.0.5] saw for disclosing of [0387.0.0.5] to [0392.0.0.5] these paragraphs

[0393.0.5.5] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 63-67 and 436-449 is capable, preferred Table I B the 5th or 7 row, 63-67 and the 436-449 nucleic acid molecule shown in capable is hybridized under lax stringent condition nucleic acid molecule, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

Paragraph [0394.0.0.0] was to [0416.0.0.0] above [0394.0.0.5] to [0415.0.0.5] and [0416.0.5.5] saw for disclosing of [0394.0.0.5] to [0415.0.0.5] and [0416.0.5.5] these paragraphs

[0417.0.5.5] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of fatty acid biosynthetic pathway is had resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block lipid acid in the described biology (particularly fine chemicals) synthetic inhibitor.The example of lipid acid synthetic inhibitor or weedicide is for suppressing the cerulenin of lipid acid (beta-keto acyl base thioesters synthetase inhibitors) in the blocking-up biological (for example microorganism or plant); Thiolactomycin; Diazoborines or trichlorophenol and sterol biosynthesis inhibitor be chloroformate grass oxazole diclofop-methyl for example; the spirit of pyrrole fluorine chlorine standing grain; fluazifop or quizalofop; the cyclohexyl diketone that perhaps suppresses plant acetyl coenzyme A carboxylase is clethodim or sethoxydim [(2-[1-{ ethoxy imino } butyl]-5-[2-{ ethylmercapto group for example } propyl group]-3-hydroxyl-2-tetrahydrobenzene-1-ketone), perhaps thiocarbamate butylate for example; EPTC[=S-ethyl dipropyl thiol carbamate] or vernolate.

Paragraph [0418.0.0.0] was to [0430.0.0.0] above [0418.0.0.5] to [0430.0.0.5] saw for disclosing of [0418.0.0.5] to [0430.0.0.5] these paragraphs

[0431.0.5.5], [0432.0.5.5], [0433.0.0.5] and [0434.0.0.5] be for [0431.0.5.5],

[0432.0.5.5], disclosing of [0433.0.0.5] and [0434.0.0.5] these paragraphs sees that top paragraph [0431.0.0.0] is to [0434.0.0.0]

[0435.0.5.5] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.5.5] can change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce lipid acid.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coliand Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces the expection fine chemicals by screening can screen positive catastrophic event.

[0437.0.5.5] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.5.5] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.5.5] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold Spring HarborLab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical Tips Online1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.5.5] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

Above [0441.0.0.5] sees for disclosing of this paragraph [0441.0.0.0]

Suddenly change in [0442.0.5.5] observation transformed host cell or transgenic protein activity reaches this fact in the mode similar to wild-type protein with similar scale based on protein.Measuring appropriate method that mutant gene or transgenosis transcribe quantity (can for the sign of the mRNA quantity of translation gene product) (consults as Ausubel etc. for carrying out the Northern trace, (1988) " Current Protocols inMolecular Biology ", Wiley:New York), wherein provide primer to design with goal gene bonded mode with detectable mark (being generally radioactivity or chemiluminescent labeling), thereby as the total RNA that extracts the biological culture thing, in glue, separate, be applied to stable matrix and when hatching, the combination of probe and indicate the existence and the quantity of this gene mRNA in conjunction with quantity with this probe.Other method is a quantitative PCR.This information detects the degree of genetic transcription.Can use several different methods known in the art, for example use the Ambion test kit and according to the specification sheets of manufacturers, perhaps as Edgington etc., Promega Notes Magazine Number 41,1993, the 14 pages described separates total cell RNA from for example yeast or intestinal bacteria.

Above [0443.0.0.5] sees for disclosing of this paragraph [0443.0.0.0]

[0444.0.5.5] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.5.5] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

Paragraph [0446.0.0.0] was to [0453.0.0.0] above [0446.0.0.5] to [0453.0.0.5] saw for disclosing of [0446.0.0.5] to [0453.0.0.5] these paragraphs

[0454.0.5.5] embodiment 8: the influence that analyzing nucleic acid molecules produces lipid acid

[0455.0.5.5] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example lipid acid) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being lipid or lipid acid) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

Except said process, as (1999) Proc.Natl.Acad.Sci.USA96 (22) such as Cahoon: from vegetable material, extract plant lipid as described in (1986) Analytic Biochemistry152:141-145 such as 12935-12940 and Browse.The qualitative and quantitative analysis of lipid or lipid acid is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:Oily Press (Oily Press Lipid Library; 2); Christie, WilliamW., Gas Chromatography and Lipids.A Practical Guide-Ayr, Scotland:Oily Press, 1989, Repr.1992, IX, 307pp. (Oily Press Lipid Library; 1); " Progress in Lipid Research, Oxford:Pergamon Press, 1 (1952)-16 (1977), exercise question is Progress in the Chemistry of Fats and Other Lipids CODEN.

Above [0456.0.0.5] sees for disclosing of this paragraph [0456.0.0.0]

[0457.0.5.5] embodiment 9: the purifying of lipid acid

[0458.0.5.5] embodiment analyzes lipid acid (abbreviation: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatograph/mass spectrum; TAG, triacylglycerol; TLC, thin-layer chromatography).By using standard method of analysis GC, GC-MS or TLC can clearly detect the existence of fatty acids products to the biology analysis of recombinating, described analytical procedure Christie and reference wherein are described (1997, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric method], Lipide 33:343-353).The biological total fatty acids that yeast produced that for example is used for the inventive method can be according to the following step analysis: by ultrasonic, the grinding of glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed such as yeast, intestinal bacteria or plant fragmentation.After the fragmentation, must material is centrifugal (1000xg, 10 minutes, 4 ℃) also use 100mM NaHCO ₃, pH 8.0 washings are once to remove residual substratum and lipid acid.In order to prepare fatty acid methyl ester (FAMES), with pellet resuspended in distilled water, in 100 ℃ of heating 10 minutes, cooled on ice is also centrifugal, then in containing the 0.5M sulfuric acid (in methyl alcohol) of 2% Propanal dimethyl acetal, extracted 1 hour in 90 ℃, this causes producing the oil and the fat compound of hydrolysis, and this can obtain transmethylase fat.Then, use 2ml Petroleum ether extraction FAMES twice, use 100mM NaHCO then ₃, pH 8.0 washs once and uses Na ₂SO ₄Dry.Under argon gas stream,, and FAMES heavily is dissolved in the 50 μ l sherwood oils organic solvent evaporation.ZEBRON ZB-Wax capillary column in having Hewlett Packard 6850 gas chromatographs of flame ionization detector (30m, 0,32mm, 0,25 μ m; Phenomenex) go up sample separation.Furnace temperature is carried out time variable control, with 20 ℃/minute speed from 70 ℃ (keeping 1 minute) to 200 ℃, then with 5 ℃/minute speed to 250 ℃ (keeping 5 minutes), at last with 5 ℃/minute speed to 260 ℃.Nitrogen is as carrier gas (4.5ml/ minute, under 70 ℃).The identity of resulting fatty acid methyl ester must be used and can define from the standard that commercial source (being Sigma) obtains.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Then, in 100 ℃ of heating 10 minutes, centrifugation again after the cooled on ice.With cell precipitation in 1M sulfuric acid methanol solution and 2% Propanal dimethyl acetal in 90 ℃ of hydrolysis 1 hour, lipid is by transmethylase.Resulting fatty acid methyl ester (FAME) extracts in sherwood oil.The FAME that is extracted by use capillary column (Chrompack, the WCOT fused silica, CP-Wax-52 CB, 25 μ m 0.32mm) carried out gas chromatographic analysis in following 5 minutes in 170 ℃-240 ℃ gradient temperature 20 minutes and 240 ℃.The identity of fatty acid methyl ester must be by relatively confirming with corresponding FAME (being Sigma).Can be suitable chemically derived by the FAME mixture is carried out, for example obtain 4 by GC-MS, 4-Er Jia Yang oxazolin derivs (Christie, 1998) is further identified identity and position of double bond.

Method is described in as Napier and Michaelson, and 2001, Lipids.36 (8): 761-766; Sayanova etc., 2001, Journal of Experimental Botany.52 (360): 1581-1585, Sperling etc., 2001, Arch.Biochem.Biophys.388 (2): 293-298 and Michaelson etc., 1998, FEBS Letters.439 (3): among the 215-218.

[0459.0.5.5] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified lipid acid.Because of the elutriant difference, can advantageously use acetonitrile/water and chloroform/acetonitrile mixture.For example, (this HPLC method Germany) has been separated rapeseed oil for ET 250/3 Nucleosil 120-5 C18 Macherey und Nagel, D ü ren by using the RP-18-post.Chloroform/acetonitrile mixture (v/v 30: 70) is as elutriant.0.8ml/ minute flow velocity be favourable.In order to analyze lipid acid, used ELSD detector (light scattering detector).For the purifying of lipid acid, MPLC, dried post flash chromatography (dry-flash chromatography) or thin-layer chromatography are other favourable chromatographic processes.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.5.5] depends on the fine chemicals that is produced in addition, can also use crystallization and distillation to come purifying.These two kinds of methods are that those skilled in the art are well-known.

[0461.0.5.5] embodiment 10: clone SEQ ID NO:4464 is used for expressing plant

Above [0462.0.0.5] sees for disclosing of this paragraph [0462.0.0.0]

[0463.0.5.5] passes through pcr amplification SEQ ID NO:4464 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

The composition of [0464.0.5.5] Pfu Turbo archaeal dna polymerase schedule of operation is as follows: 1x PCR damping fluid (Stratagene), every kind of dNTP of 0.2mM, 100ng yeast saccharomyces cerevisiae (bacterial strain S288C; Research Genetics, Inc. now is Invitrogen) or intestinal bacteria (bacterial strain MG1655; E.coli Genetic Stock Center) genomic dna, 50pmol forward primer, 50pmol reverse primer, 2.5u Pfu Turbo archaeal dna polymerase.Amplification cycles is as follows:

[0465.0.5.5] 94-95 ℃ of circulation in 3 minutes, 95 ℃ of 1 minute or 94 ℃ 30 seconds, 50 ℃ 45 seconds, 50 ℃ 30 seconds or 55 ℃ 30 seconds and 72 ℃ of 210-480 seconds then, 28-36 circulation under each situation, 72 ℃ of circulations in 8 minutes then, 4 ℃ then.

Above [0466.0.0.5] sees for disclosing of this paragraph [0466.0.0.0]

[0467.0.5.5] selects following primer sequence for genes of SEQ ID NO:4464:

I) forward primer (SEQ ID NO:4556)

atgggacaca?agcccttata?ccg

Ii) reverse primer (SEQ ID NO:4557)

ttatcgcgat?gattttcgct?gcg

Paragraph [0468.0.0.0] was to [0479.0.0.0] above [0468.0.0.5] to [0479.0.0.5] saw for disclosing of [0468.0.0.5] to [0479.0.0.5] these paragraphs

[0480.0.5.5] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:4464

Above [0481.0.0.5] sees for disclosing of this paragraph [0481.0.0.0]

Paragraph [0482.0.0.0] was to [0513.0.0.0] above [0482.0.0.5] to [0513.0.0.5] saw for disclosing of [0482.0.0.5] to [0513.0.0.5] these paragraphs

[0514.0.5.5] selects as another kind of, as people such as Geigenberger (Plant Cell﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect lipid acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??b0730	Linolic acid (C18:2 (c9, c12))	??GC	??1.13	??1.34
??b3256	Linolic acid (C18:2 (c9, c12))	??GC	??1.13	??1.20
					??YBR089C-A	Linolic acid (C18:2 (c9, c12))	??GC	??1.36	??1.70

??YDR447C	Linolic acid (C18:2 (c9, c12))	??GC	??1.18	??2.40
					??YOR024W	Linolic acid (C18:2 (c9, c12))	??GC	??1.16	??1.33
??b0050	Linolic acid (C18 :) along [9,12] 2	??GC	??1.15	??1.37
					??b0251	Linolic acid (C18 :) along [9,12] 2	??GC	??1.14	??1.31
??b0255	Linolic acid (C18 :) along [9,12] 2	??GC	??1.22	??1.31
					??b0577	Linolic acid (C18 :) along [9,12] 2	??GC	??1.17	??1.40
??b0849	Linolic acid (C18 :) along [9,12] 2	??GC	??1.19	??1.34
					??b1097	Linolic acid (C18 :) along [9,12] 2	??GC	??1.15	??1.30
??b1693	Linolic acid (C18 :) along [9,12] 2	??GC	??1.17	??1.32
					??b2710	Linolic acid (C18 :) along [9,12] 2	??GC	??1.15	??1.29
??b2822	Linolic acid (C18 :) along [9,12] 2	??GC	??1.15	??1.27
					??b3064	Linolic acid (C18 :) along [9,12] 2	??GC	??1.17	??1.30
??b3166	Linolic acid (C18 :) along [9,12] 2	??GC	??1.14	??1.21
					??b3457	Linolic acid (C18 :) along [9,12] 2	??GC	??1.20	??1.44
??b3644	Linolic acid (C18 :) along [9,12] 2	??GC	??1.14	??1.35
					??b4129	Linolic acid (C18 :) along [9,12] 2	??GC	??1.20	??1.47

[0515.0.5.5] the 2nd row have shown the lipid acid of being analyzed.The 4th row and the 5th row have shown the ratio of lipid acid between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

[0516.0.0.5] and [0517.0.5.5] sees top paragraph [0516.0.0.0] and [0517.0.0.0] for disclosing of [0516.0.0.5] and [0517.0.5.5] these paragraphs

Paragraph [0518.0.0.0] was to [0530.0.0.0] above [0518.0.0.5] to [0529.0.0.5] and [0530.0.5.5] saw for disclosing of [0518.0.0.5] to [0529.0.0.5] and [0530.0.5.5] these paragraphs

Paragraph [0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.5] to [0530.6.0.5] saw for disclosing of [0530.1.0.5] to [0530.6.0.5] these paragraphs

Paragraph [0531.0.0.0] was to [0534.0.0.0] above [0531.0.0.5] to [0533.0.0.5] and [0534.0.5.5] saw for disclosing of [0531.0.0.5] to [0533.0.0.5] and [0534.0.5.5] these paragraphs

Paragraph [0535.0.0.0] was to [0538.0.0.0] above [0535.0.0.5] to [0537.0.0.5] and [0538.0.5.5] saw for disclosing of [0535.0.0.5] to [0537.0.0.5] and [0538.0.5.5] these paragraphs

Paragraph [0539.0.0.0] was to [0543.0.0.0] above [0539.0.0.5] to [0542.0.0.5] and [0543.0.5.5] saw for disclosing of [0539.0.0.5] to [0542.0.0.5] and [0543.0.5.5] these paragraphs

Paragraph above [0544.0.0.5] to [0547.0.0.5] and [0548.0.5.5] see for disclosing of [0544.0.0.5] to [0547.0.0.5] and [0548.0.5.5] to [0552.0.0.5] these paragraphs to [0552.0.0.5]

[0544.0.0.0] is to [0552.0.0.0]

Above [0552.2.0.5] sees for disclosing of this paragraph [0552.2.0.0]

[0553.0.5.5]

1. produce linoleic method, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable or its function equivalent; With

(b) in allowing described biology, produce this biology of cultivation under the linoleic condition.

2. produce linoleic method, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable, and described nucleic acid molecule gives that linolic acid quantity increases in biology or its part;

B) contain just like Table I the 5th or 7 row, 63-67 and 436-449 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of linolic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of linolic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that linolic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 63-67 and 436-449 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that linolic acid quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that linolic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 63-67 and 436-449 capable shown in consensus sequence polypeptide and give biology or its part in linolic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give linolic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded linolic acid.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded linolic acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 63-67 and 436-449 are capable, and described nucleic acid molecule gives that linolic acid quantity increases in biology or its part;

B) contain just like Table I the 5th or 7 row, 63-67 and 436-449 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of linolic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of linolic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that linolic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 5th or 7 row, 63-67 and 436-449 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that each fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that linolic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 63-67 and 436-449 capable shown in consensus sequence polypeptide and give biology or its part in linolic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give linolic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A the 5th or 7 row, 63-67 and 436-449 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 63-67 and 436-449 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in linolic acid quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part linolic acid quantity increase coded by the nucleic acid molecule of claim 5 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps linolic acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the linolic acid level that will measure or expression of polypeptides level and standard linolic acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the linolic acid generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of linolic acid quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of linolic acid quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the linolic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that linolic acid increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the suitable linoleic host cell that produces;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) the linolic acid level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give linolic acid level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the linolic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that linolic acid quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the suitable linoleic host cell that produces;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) the linolic acid level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give linolic acid level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that linolic acid increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control linolic acid level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make the anti-linolic acid synthetic weedicide that suppresses of plant.

[0554.0.0.5] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.6] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.6] sees [0001.0.0.0] for disclosing of this section.

Above [0002.0.5.6] to [0008.0.5.6] sees for disclosing of [0002.0.5.6] to [0008.0.5.6] these paragraphs [0002.0.0.0] and [0008.0.0.0]

[0009.0.6.6] as mentioned above, indispensable fatty acid is that people and many Mammalss such as domestic animal are necessary.Indispensable fatty acid such as alpha-linolenic acid are for rehabilitation and to keep good condition of health be extremely important.Verified, the compound that produces from alpha-linolenic acid can reduce the blood clotting and the inflammatory process of health.

Above [0010.0.6.6] to [0012.0.6.6] sees for disclosing of [0010.0.6.6] to [0012.0.6.6] these paragraphs [0010.0.0.0] and [0012.0.0.0]

[0013.0.0.6] sees [0013.0.0.0] for disclosing of this paragraph

[0014.0.6.6] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is alpha-linolenic acid or the triglyceride level that contains alpha-linolenic acid, lipid, oils or fat.Therefore, in the present invention, term " fine chemicals " is meant " alpha-linolenic acid and/or contain triglyceride level, lipid, oils or the fat of alpha-linolenic acid " as used herein.In addition, term " fine chemicals " also refers to comprise alpha-linolenic acid and/or contains the triglyceride level, lipid, oils of alpha-linolenic acid or the fine chemicals of fat as used herein.

[0015.0.6.6] an embodiment, term " fine chemicals " meaning is meant alpha-linolenic acid and/or contains triglyceride level, lipid, oils or the fat of alpha-linolenic acid.In whole specification sheets, term " fine chemicals " meaning is meant alpha-linolenic acid and/or contains triglyceride level, lipid, oils or fat, alpha-linolenic acid or its salt, ester, thioesters or the free form of alpha-linolenic acid or in conjunction with the alpha-linolenic acid of other compound such as triacylglycerol, glycolipid, phosphatide etc.In preferred embodiments, term " fine chemicals " meaning is meant free alpha-linolenic acid or its salt or is bonded to the alpha-linolenic acid of triacylglycerol.Triacylglycerol, lipid, oils, fat or its lipid mixtures should refer to any triacylglycerol, lipid, oils and/or contain the fat of any combination or free alpha-linolenic acid, for example sphingolipid, phosphoglyceride, lipid, glycolipid class such as sphingoglycolipid, phosphatide such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, glycerine Serine, phosphatidylinositols or two glycerine Serines or monoacylglycerol, diacylglycerol or triacylglycerol or other fatty acid ester such as acetyl-CoA thioesters, it contains other saturated or unsaturated fatty acids in fatty acid molecule.

[0016.0.6.6] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce protein b2699, YER173W, YGL205W, YIL150C, b0050, b0251, b0376, b0577, b0849, b2822, b3129, b3457, b3462 and/or b3644 or have by Table I the 5th or 7 row, 68-71 and 450-459 capable shown in the activity of proteins of the coded peptide sequence of nucleic acid molecule; With

(b) in allowing described biology, produce under the condition that fine chemicals is alpha-linolenic acid or the fine chemicals that contains alpha-linolenic acid and make biological growth.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II the 3rd row, 68-71 and 450-459 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, 68-71 and 450-459 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) under the condition that allows generation fine chemicals, particularly alpha-linolenic acid, make biological growth.

Above [0017.0.0.6] and [0018.0.0.6] sees for disclosing of [0017.0.0.6] and [0018.0.0.6] these paragraphs [0017.0.0.0] and [0018.0.0.0]

The method that [0019.0.6.6] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare the fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 68-71 and 450-459 are capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 68-71 and 450-459 were capable.

[0020.0.6.6] be surprisingly found out that, at least a as the yeast saccharomyces cerevisiae protein shown in Table II the 3rd row, 69-71 are capable and/or at least a such as Table II the 3rd row, the 68th and e. coli k12 protein in Arabidopis thaliana the transgene expression of 450-459 shown in capable give institute's conversion plant alpha-linolenic acid (or fine chemicals) content raising.

[0021.0.0.6] sees [0021.0.0.0] for disclosing of this paragraph

The sequence of [0022.0.6.6] e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the auxilliary proteolytic enzyme of DNA dependency ATP enzyme or DNA and ATP dependency.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary DNA dependency ATP enzyme or DNA and the auxilliary proteolytic enzyme of ATP dependency or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the auxilliary protease activities of DNA dependency ATP enzyme or DNA and ATP dependency is enhanced or produces in the methods of the invention, and for example the activity from colibacillary DNA dependency ATP enzyme or DNA and the auxilliary proteolytic enzyme of ATP dependency or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YER173W has been published in Dietrich etc., Nature 387 (6632 supplementary issue), and 78-81 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.It seemingly " participates in dna damage and reduction division pachytene stage check position activatory check position protein; Rad17p-Mec3p-Ddc1p is loaded into the subunit of the clip loading bin of DNA, and it may be people and the proteinic homologue of schizosaccharomyces pombe Rad17 ".Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YER173W from yeast saccharomyces cerevisiae as shown here, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the YER173W activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YER173W protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YGL205W has been published in Tettelin etc.; Nature 387 (6632 supplementary issue); 81-84 (1997) and Goffeau etc.; Science 274 (5287); 546-547; 1996, and its activity to be defined as be " ACOD that is positioned at the participation lipid acid β-Yang Hua approach of peroxysome ".Therefore; in one embodiment; the inventive method comprises " ACOD " as shown here or the purposes of its homologue; as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the activity of ACOD is enhanced or produces in the methods of the invention, for example is enhanced or produces from the ACOD of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Churcher etc., Nature 387 (6632 supplementary issue), 84-87 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be defined as be " S phase (DNA synthetic) initial or finish necessary chromobindins ".Therefore, in one embodiment, the inventive method comprises the purposes from yeast saccharomyces cerevisiae chromobindins or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the activity of chromobindins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the chromobindins of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0050 (accession number NP_414592) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be potential participation protein-protein interaction conservative protein matter.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary described protein or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, described in the methods of the invention activity of proteins is enhanced or produces, and for example the activity from colibacillary described protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b0251 (accession number NP_414785) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition HTH-type transcriptional.Therefore, in one embodiment, the inventive method comprises as shown here from the HTH-type transcription regulatory protein of colibacillary supposition or the purposes of its homologue, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the activity of Jia Ding HTH-type transcription regulatory protein is enhanced or produces in the methods of the invention, and for example HTH-type transcription regulatory protein or its homologue activity from colibacillary supposition is enhanced or produces.

The sequence of e. coli k12 b0376 (accession number NP_414910) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be protein β-Nei Xiananmei/D-alanine carboxypeptidase in conjunction with penicillin.Therefore, in one embodiment, the inventive method comprise as shown here from colibacillary in conjunction with the protein β-Nei Xiananmei/D-alanine carboxypeptidase of penicillin or the purposes of its homologue, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, activity in conjunction with the protein β-Nei Xiananmei/D-alanine carboxypeptidase of penicillin is enhanced or produces in the methods of the invention, for example is enhanced or produces in conjunction with the protein β-Nei Xiananmei/D-alanine carboxypeptidase of penicillin or the activity of its homologue from colibacillary.

The sequence of e. coli k12 b0577 (accession number NP_415109) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition translocator.Therefore, in one embodiment, the inventive method comprises the purposes of translocator or its homologue from colibacillary supposition as shown here, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the activity of Jia Ding translocator is enhanced or produces in the methods of the invention, for example is enhanced or produces from the translocator of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b0849 (accession number NP_415370) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase.Therefore, in one embodiment, the inventive method comprises as shown here from the glutaredoxin 1 redox cofactor protein of colibacillary gsh dependency ribonucleotide reductase or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat particularly increase alpha-linolenic acid and/or contain the triglyceride level of alpha-linolenic acid, lipid, oils and/or fat, the quantity of the alpha-linolenic acid of preferred free or combining form.In one embodiment, the activity of the glutaredoxin 1 redox cofactor protein of gsh dependency ribonucleotide reductase is enhanced or produces in the methods of the invention, for example is enhanced or produces from the glutaredoxin 1 redox cofactor protein of colibacillary gsh dependency ribonucleotide reductase or the activity of its homologue.

The sequence of e. coli k12 b2822 (accession number NP_417299) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease " that its activity is defined as.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease zymoprotein " or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat particularly increase alpha-linolenic acid and/or contain the triglyceride level of alpha-linolenic acid, lipid, oils and/or fat, the quantity of the alpha-linolenic acid of preferred free or combining form.In one embodiment, the activity of " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease zymoprotein " is enhanced or produces in the methods of the invention, for example be enhanced or produce from colibacillary " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease zymoprotein " or the activity of its homologue.

The sequence of e. coli k12 b3129 (accession number NP_417598) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be " the proteolytic enzyme of supposition; HtrA repressor ".Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary " proteolytic enzyme of supposition; The htrA repressor " or the purposes of its homologue; as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form." the proteolytic enzyme of supposition in the methods of the invention in one embodiment; The htrA repressor " activity be enhanced or produce, for example from the colibacillary " proteolytic enzyme of supposition; The htrA repressor " or the activity of its homologue be enhanced or produce.

The sequence of e. coli k12 b3457 (accession number NP_417914) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary high-affinity branched-chain amino acid translocator or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the activity of high-affinity branched-chain amino acid translocator is enhanced or produces in the methods of the invention, and for example the activity from colibacillary high-affinity branched-chain amino acid translocator or its homologue is enhanced or produces.

The sequence of e. coli k12 b3462 (accession number NP_417919) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the conformity membrane cell division protein.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary conformity membrane cell division protein or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the activity of conformity membrane cell division protein is enhanced or produces in the methods of the invention, and for example the activity from colibacillary conformity membrane cell division protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b3644 (accession number NP_418101) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the stress-induced protein that does not characterize.Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary stress-induced protein that does not characterize or the purposes of its homologue, as described its be used for biological or its part produces fine chemicals, be alpha-linolenic acid and/or the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat, particularly increase alpha-linolenic acid and/or contain triglyceride level, lipid, oils and/or the fat of alpha-linolenic acid, the preferred quantity of the alpha-linolenic acid of free or combining form.In one embodiment, the stress-induced activity of proteins that does not characterize is enhanced or produces in the methods of the invention, for example is enhanced or produces from the colibacillary stress-induced protein that does not characterize or the activity of its homologue.

The homologous compound (=homologue) of [0023.0.6.6] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.

Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with protein shown at least a Table II the 3rd row, 68-71 and 450-459 are capable, for example have by contain Table I the 5th or 7 row, 68-71 and 450-459 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in Table II 69-71 is capable in the polypeptide homologue of any one be to have a same or similar active homologue, particularly active increasing gives that fine chemicals content increases in the biology, and described homologue is from eukaryote.In one embodiment, Table II the 3rd row, the 68th and 450-459 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 69-71 are capable is to have a same or similar active homologue, and particularly active increasing gives that fine chemicals content increases in biology or its part, and described homologue is from fungi.In one embodiment, Table II the 3rd row, the 68th and 450-459 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Proteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 69-71 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Ascomycota.In one embodiment, Table II the 3rd row, the 68th and 450-459 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 69-71 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from yeast.In one embodiment, Table II the 3rd row, the 68th and 450-459 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the enterobacteria order.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 69-71 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.In one embodiment, Table II the 3rd row, the 68th and 450-459 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from enterobacteriaceae.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 69-71 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetes.In one embodiment, Table II the 3rd row, the 68th and 450-459 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Escherichia.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 69-71 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetaceae.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 69-71 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.

Above [0023.1.0.6] and [0024.0.0.6] sees for disclosing of [0023.1.0.6] and [0024.0.0.6] these paragraphs [0023.1.0.0] and [0024.0.0.0]

[0025.0.6.6] is according to the present invention, if triglyceride level, lipid, oils or fat level that from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause alpha-linolenic acid in biology or its part, the preferred described biomass cells and/or contain alpha-linolenic acid increase and protein has as proteinic above-mentioned activity shown in Table II the 3rd row, 68-71 and 450-459 are capable, then described protein or polypeptide have " as activity of proteins shown in Table II the 3rd row, 68-71 and 450-459 are capable ".In this manual, if this kind protein or polypeptide still have Table II the 3rd row, proteinic biology or the enzyme activity shown in 68-71 and 450-459 are capable, if promptly with Table II the 3rd row, yeast saccharomyces cerevisiae protein shown in 69-71 is capable is compared and/or is listed as with Table II the 3rd, the 68th compares with e. coli k12 protein shown in 450-459 is capable, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

Above [0025.1.0.6] and [0025.2.0.6] sees for disclosing of [0025.1.0.6] and [0025.2.0.6] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.6] to [0033.0.0.6] saw for disclosing of [0026.0.0.6] to [0033.0.0.6] these paragraphs

[0034.0.6.6] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 68-71 and protein shown in 450-459 is capable or by the active protein expression level of the protein of Table I the 5th row, 68-71 and nucleic acid molecule encoding shown in 450-459 is capable or its homologue (as Table I the 7th row, 68-71 and homologue shown in 450-459 is capable) or active aspect different, and it is difference aspect biological chemistry or genetics reason, and therefore shows the fine chemicals quantity that increases.

[0035.0.0.0] was to [0039.0.0.0] above [0035.0.0.6] to [0038.0.0.6] and [0039.0.5.6] saw for disclosing of [0035.0.0.6] to [0038.0.0.6] and [0039.0.5.6] these paragraphs

Above [0040.0.0.6] to [0044.0.0.6] sees for disclosing of [0040.0.0.6] to [0044.0.0.6] these paragraphs [0035.0.0.0] and [0044.0.0.0]

[0045.0.6.6] in one embodiment, at e. coli k12 protein b2699 or its homologue, for example under the situation that the activity of the auxilliary protease protein matter of DNA dependency ATP enzyme or DNA and ATP dependency (for example shown in Table II the 5th or 7 row, the 68th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 13% and 32% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example " not profiling protein matter YER173W " (it seemingly " participates in dna damage and reduction division pachytene stage check position activatory check position protein; Rad17p-Mec3p-Ddc1p is loaded into the subunit of the clip loading bin of DNA.It may be people and the proteinic homologue of schizosaccharomyces pombe Rad17 ") under the activity situation about being enhanced of (for example shown in Table II the 5th or 7 row, the 69th row); preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 15% and 54% or more between.

In one embodiment; at yeast saccharomyces cerevisiae protein YGL205W or its homologue; for example under the activity situation about being enhanced of ACOD protein (for example shown in Table II the 5th or 7 row, the 70th row); preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 13% and 24% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, under the activity situation about being enhanced of for example " chromobindins " (for example shown in Table II the 5th or 7 row, the 71st row), preferably, in one embodiment, give fine chemicals, preferred alpha-linolenic acid increase at least 243%.

At e. coli k12 protein b0050 or its homologue, under the activity situation about being enhanced of for example potential participation protein-protein interaction conservative protein matter (for example shown in Table II the 5th or 7 row, the 450th row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 11% and 19% or more between.

At e. coli k12 protein b0251 or its homologue, for example under the activity situation about being enhanced of Jia Ding HTH-type transcriptional (for example shown in Table II the 5th or 7 row, the 451st row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 14% and 29% or more between.

At e. coli k12 protein b0376 or its homologue, for example under the situation about being enhanced in conjunction with the activity of the protein β-Nei Xiananmei/D-alanine carboxypeptidase of penicillin (for example shown in Table II the 5th or 7 row, the 452nd row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 15% and 34% or more between.

At e. coli k12 protein b0577 or its homologue, for example under the activity situation about being enhanced of Jia Ding translocator (for example shown in Table II the 5th or 7 row, the 453rd row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 12% and 96% or more between.

At e. coli k12 protein b0849 or its homologue, for example under the activity situation about being enhanced of the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase (for example shown in Table II the 5th or 7 row, the 454th row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 13% and 31% or more between.

At e. coli k12 protein b2822 or its homologue, under the activity situation about being enhanced of for example " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease " (for example shown in Table II the 5th or 7 row, the 455th row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 15% and 20% or more between.

At e. coli k12 protein b3129 or its homologue, for example " proteolytic enzyme of supposition; The htrA repressor " under the activity situation about being enhanced of (for example shown in Table II the 5th or 7 row, the 456th row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 12% and 26% or more between.

At e. coli k12 protein b3457 or its homologue, for example under the activity situation about being enhanced of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) (for example shown in Table II the 5th or 7 row, the 457th row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 15% and 28% or more between.

At e. coli k12 protein b3462 or its homologue, for example under the activity situation about being enhanced of conformity membrane cell division protein (for example shown in Table II the 5th or 7 row, the 458th row), preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 13% and 17% or more between.

At e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 459th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred alpha-linolenic acid be increased in 13% and 36% or more between.

[0046.0.6.6] is at e. coli k12 protein b2699 or its homologue, for example under the situation that the activity of the auxilliary protease protein matter of DNA dependency ATP enzyme or DNA and ATP dependency is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

At yeast saccharomyces cerevisiae protein YER173W or its homologue, for example under the situation that the activity of " not profiling protein matter YER173W " is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

At yeast saccharomyces cerevisiae protein YGL205W or its homologue, for example under the situation that the ACOD activity of proteins is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

At yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example under the situation that the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " is enhanced, triglyceride level, lipid, oils and/or the fat preferably giving fine chemicals and contain alpha-linolenic acid increase.

In one embodiment, at e. coli k12 protein b0050 or its homologue, under the situation that the activity of for example potential participation protein-protein interaction conservative protein matter is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b0251 or its homologue, for example under the situation that the activity of Jia Ding HTH-type transcriptional is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b0376 or its homologue, for example under the situation about being enhanced in conjunction with the activity of the protein β-Nei Xiananmei/D-alanine carboxypeptidase of penicillin, triglyceride level, lipid, oils and/or the fat preferably giving fine chemicals and contain alpha-linolenic acid increase.

In one embodiment, at e. coli k12 protein b0577 or its homologue, for example under the situation that the activity of Jia Ding translocator is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b0849 or its homologue, for example under the situation that the activity of the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b2822 or its homologue, for example under the situation that the activity of " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease " is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b3129 or its homologue, for example " proteolytic enzyme of supposition; The htrA repressor " activity situation about being enhanced under, triglyceride level, lipid, oils and/or the fat preferably giving fine chemicals and contain alpha-linolenic acid increase.

In one embodiment, at e. coli k12 protein b3457 or its homologue, for example under the situation that the activity of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b3462 or its homologue, for example under the situation that the activity of conformity membrane cell division protein is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b3644 or its homologue, for example under the situation that the stress-induced activity of proteins that does not characterize is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains alpha-linolenic acid, lipid, oils and/or fat.

Above [0047.0.0.6] to [0048.0.0.6] sees for disclosing of [0047.0.0.6] and [0048.0.0.6] these paragraphs [0047.0.0.0] and [0048.0.0.0]

[0049.0.6.6] has to give and improves each fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, the polypeptide of consensus sequence shown in 68-71 and 450-459 are capable, perhaps as Table II the 5th and 7 row, the polypeptide of aminoacid sequence shown in 68-71 and 450-459 are capable or its function homologue described in the literary composition, perhaps (for example be listed as by Table I the 5th and 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 68-71 and 450-459 are capable or its function homologue as herein described) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.6.6] for the purposes of the present invention, term " alpha-linolenic acid " also comprises corresponding salt, for example the sylvite of alpha-linolenic acid or sodium salt or have amine such as the salt of the alpha-linolenic acid of diethylamine.

Above [0051.0.5.6] and [0052.0.0.6] sees for disclosing of [0051.0.5.6] and [0052.0.0.6] these paragraphs [0051.0.0.0] and [0052.0.0.0]

[0053.0.6.6] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having as the active polypeptide of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 7th row, 63-67 and 436-449 capable shown in)) and is expressed and increase, and has the activity of the raising fine chemicals described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example having as the active polypeptide of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 7th row, 68-71 and 450-459 capable shown in)) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of the raising alpha-linolenic acid described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give the raising alpha-linolenic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having) as the active polypeptide of protein or its homologue shown in Table II the 3rd row, 68-71 and 450-459 are capable (for example as Table II the 7th row, 68-71 and 450-459 are capable shown in), perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give the raising alpha-linolenic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 7th row, 68-71 and 450-459 capable shown in);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give the raising alpha-linolenic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 7th row, 68-71 and 450-459 capable shown in);

(f) express the transgenosis of coded protein, described protein give the raising alpha-linolenic acid that has described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 7th row, 68-71 and 450-459 capable shown in);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have the raising alpha-linolenic acid described in the literary composition active, by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 7th row, 68-71 and 450-459 capable shown in);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide or the used polypeptide of the inventive method (for example having as the active polypeptide of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 7th row, 68-71 and 450-459 capable shown in)).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.6.6] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example after active expression of polypeptides of protein shown in Table II the 3rd row, 68-71 and 450-459 are capable or its homologue (for example Table II the 5th or 7 row, 68-71 and 450-459 capable shown in polypeptide) or activity, give alpha-linolenic acid and increase improving coded protein or have.

[0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.6] to [0067.0.0.6] saw for disclosing of [0055.0.0.6] to [0067.0.0.6] these paragraphs

Above [0068.0.5.6] and [0069.0.5.6] sees for disclosing of [0068.0.5.6] and [0069.0.5.6] these paragraphs [0068.0.0.0] and [0069.0.0.0]

Above [0070.0.6.6] and [0071.0.5.6] sees for disclosing of [0070.0.6.6] and [0071.0.5.6] these paragraphs [0070.0.5.5] and [0071.0.0.0]

[0072.0.6.6] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has other compound, for example palmitinic acid, Zoomeric acid, stearic acid, oleic acid and/or linolic acid except alpha-linolenic acid, the triacylglycerol that contains alpha-linolenic acid, lipid, oils and/or fat.

[0073.0.6.6] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of protein with polypeptide of the present invention or the used polypeptide active of the inventive method or its homologue (for example Table II the 5th or 7 row, 68-71 and 450-459 capable shown in) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of promptly giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and other optional free and/or conjugated fatty acid.

[0074.0.5.6] sees [0074.0.5.5] for disclosing of this paragraph

[0075.0.0.0] was to [0084.0.0.0] above [0075.0.0.6] to [0084.0.0.6] saw for disclosing of [0075.0.0.6] to [0084.0.0.6] these paragraphs

[0085.0.6.6] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I the 5th and 7 row, 68-71 and 450-459 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as Table I the 5th and 7 row, 68-71 and nucleotide sequence or derivatives thereof shown in 450-459 is capable, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

Above [0086.0.0.6] and [0087.0.0.6] sees for disclosing of [0086.0.0.6] and [0087.0.0.6] these paragraphs [0086.0.0.0] and [0087.0.0.0]

[0088.0.6.6] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified fatty acid content.Because the nutritive value that for example is used for the plant of poultry depends on above-mentioned indispensable fatty acid and as the big scale of construction of the lipid acid in fodder energy source, so this is very important for the plant breeder.Improve or produced as Table II the 5th or 7 row, 68-71 and 450-459 capable shown in after the activity of polypeptide, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, thus the transgenic plant that produced grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0088.1.0.6], [0089.0.0.6], [0090.0.0.6] and [0091.0.5.6] is for [0088.1.0.6], [0089.0.0.6], top [0088.1.0.0] seen in disclosing of [0090.0.0.6] and [0091.0.5.6] these paragraphs, [0089.0.0.0], [0090.0.0.0] and [0091.0.0.0]

[0092.0.0.0] was to [0094.0.0.0] above [0092.0.0.6] to [0094.0.0.6] saw for disclosing of [0092.0.0.6] to [0094.0.0.6] these paragraphs

[0095.0.5.6], [0096.0.5.6] and [0097.0.5.6] are for [0095.0.5.6], and top [0095.0.5.5], [0096.0.5.5] and [0097.0.0.0] are seen in disclosing of [0096.0.5.6] and [0097.0.5.6] these paragraphs

[0098.0.6.6] in preferred embodiments, fine chemicals (alpha-linolenic acid) be produce according to the present invention and carry out isolating where necessary.It is favourable as palmitinic acid, stearic acid, Zoomeric acid, oleic acid and/or linolic acid or its mixture or other fatty acid mixt that the method according to this invention produces other lipid acid.

Above [0099.0.5.6] and [0100.0.5.6] sees for disclosing of [0099.0.5.6] and [0100.0.5.6] these paragraphs [0099.0.5.5] and [0100.0.5.5]

Above [0101.0.5.6] and [0102.0.5.6] sees for disclosing of [0101.0.5.6] and [0102.0.5.6] these paragraphs [0101.0.0.0] and [0102.0.5.5]

[0103.0.6.6] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as the sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as the sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer as sequence shown in Table III the 7th row, 68-71 and 450-459 are capable obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as the consensus sequence of sequence shown in Table IV the 7th row, 68-71 and 450-459 are capable and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, 68-71 and 450-459 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

Above [0103.1.0.6] and [0103.2.0.6] sees for disclosing of [0103.1.0.6] and [0103.2.0.6] these paragraphs [0103.1.0.0] and [0103.2.0.0]

[0104.0.6.6] in one embodiment, sequence shown in the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 68-71 and 450-459 are capable, preferably with Table I A the 5th or 7 row, 68-71 and 450-459 capable shown in sequence have one or more amino acid different, and not by Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I A the 5th or 7 row, 68-71 and 450-459 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I A the 5th or 7 row, 68-71 and 450-459 capable shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I A the 5th or 7 row, 68-71 and 450-459 capable shown in polypeptide of sequence.

Above [0105.0.0.6] to [0107.0.0.6] sees for disclosing of [0105.0.0.6] to [0107.0.0.6] these paragraphs [0105.0.0.0] and [0107.0.0.0]

Advantageously improved in [0108.0.6.6] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in 68-71 and 450-459 are capable, from Table II the 5th or 7 row, aminoacid sequence shown in 68-71 and 450-459 are capable is derived or is listed as from containing Table IV the 7th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 68-71 and 450-459 are capable, perhaps its coding has as Table II the 5th or 7 row, protein enzymic activity shown in 68-71 and 450-459 are capable or bioactive polypeptide or for example give derivative or the homologue that the fine chemicals alpha-linolenic acid increases behind its expression or active the increasing.

[0109.0.5.6] sees [0109.0.0.0] for disclosing of this paragraph

[0110.0.6.6] helps the nucleic acid molecule of the polypeptide that the inventive method and coding have the used polypeptide active of the used or of the present invention process of the inventive method (for example as protein shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or by as the protein of nucleic acid molecule encoding shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable or its homologue (shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable)) and can determine from generally open database.

[0111.0.0.6] sees [0111.0.0.0] for disclosing of this paragraph

The nucleic acid molecule that uses in [0112.0.6.6] the inventive method is the isolated nucleic acid sequences form, its coding has as the polypeptide of polypeptide active shown in Table II the 3rd row, 68-71 and 450-459 are capable or has polypeptide as peptide sequence shown in Table II the 5th and 7 row, 68-71 and 450-459 are capable, and gives each fine chemicals and increase.

Above [0113.0.0.6] to [0120.0.0.6] sees for disclosing of [0113.0.0.6] to [0120.0.0.6] these paragraphs [0113.0.0.0] and [0120.0.0.0]

[0121.0.6.6] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with the difference that one or more amino acid moleculars are arranged as peptide sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or its functional homologue as herein described, described artificial sequence is preferably given aforementioned activity, and promptly giving each fine chemicals after improving its activity increases.

Above [0122.0.0.6] to [0127.0.0.6] sees for disclosing of [0122.0.0.6] to [0127.0.0.6] these paragraphs [0122.0.0.0] and [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.6.6] polymerase chain reaction (PCR) amplification is used (for example as the primer shown in Table III the 7th row, 68-71 and 450-459 are capable) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 68-71 and 450-459 capable shown in sequence or produce as sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable.

[0129.0.6.6] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence was from described comparison shown in Table IV the 7th row, 68-71 and 450-459 were capable.

[0130.0.6.6] sees [0130.0.0.0] for disclosing of this paragraph.

[0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.6] to [0138.0.0.6] saw for disclosing of [0131.0.0.6] to [0138.0.0.6] these paragraphs

[0139.0.6.6] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 5th or 7 row, 68-71 and 450-459 capable shown in sequence hybridization, and coding is expressed and is had the active peptide of the alpha-linolenic acid of increasing.

Above [0140.0.0.6] to [0146.0.0.6] sees for disclosing of [0140.0.0.6] to [0146.0.0.6] these paragraphs [0140.0.0.0] and [0146.0.0.0]

[0147.0.6.6] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 5th or 7 row, 68-71 and 450-459 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.6.6] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 68-71 and 450-459 are capable, preferred Table I B the 5th or 7 row, nucleotide sequence shown in 68-71 and 450-459 are capable or its funtion part homology are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases fine chemicals.

[0149.0.6.6] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 5th or 7 row, 68-71 and 450-459 capable shown in one of nucleotide sequence or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (increasing as giving each fine chemicals) and as Table II the 5th or 7 row, 68-71 with 450-459 is capable, preferred Table II B the 5th or 7 row, 68-71 and the 450-459 protein shown in capable.

[00149.1.0.5] randomly, with Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 5th or 7 row, 68-71 and 450-459 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity is for being known activity or being used for these protein of note as the protein shown in Table II the 3rd row, 68-71 and 450-459 are capable.

[0150.0.6.6] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 5th or 7 row, 68-71 and 450-459 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving methionine(Met) when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 68-71 and 450-459 capable shown in sense strand, Table I the 5th or 7 row, 68-71 and the 450-459 of one of sequence capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 68-71 and 450-459 capable shown in the right PCR of primer will produce as Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 7th is listed as, 68-71 and 450-459 capable shown in the fragment of polynucleotide sequence.

[0151.0.0.6] sees [0151.0.0.0] for disclosing of this paragraph

[0152.0.6.6] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as the abundant homology of aminoacid sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable, make this albumen or its part keep to participate in produce the ability of fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising alpha-linolenic acid as be shown in the examples.

[0153.0.6.6] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as protein shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or its part have Table II the 5th for example as herein described or 7 row, 68-71 and 450-459 capable shown in the activity of polypeptide.

[0154.0.6.6] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

Above [0155.0.0.6] and [0156.0.0.6] sees for disclosing of [0155.0.0.6] and [0156.0.0.6] these paragraphs [0155.0.0.0] and [0156.0.0.0]

[0157.0.6.6] the present invention relate in addition owing to the genetic code degeneracy be different from Table I the 5th or 7 row, 68-71 and 450-459 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase) for example comprises as the polypeptide of consensus sequence shown in Table IV the 7th row, 68-71 and 450-459 are capable or as the nucleic acid molecule of polypeptide shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or its function homologue.Preferably, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as the consensus sequence shown in Table IV the 7th row, 68-71 and 450-459 are capable or as polypeptide or its function homologue shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as the consensus sequence shown in Table IV the 7th row, 68-71 and 450-459 are capable or as the basic homology of aminoacid sequence of polypeptide shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I A the 5th or 7 row, 68-71 and the 450-459 sequence shown in capable.Preferably, nucleic acid molecule of the present invention is a functional homologous compound or identical with nucleic acid molecule shown in Table I B the 5th or 7 row, 68-71 and 450-459 are capable.

[0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.6] to [0160.0.0.6] saw for disclosing of [0158.0.0.6] to [0160.0.0.6] these paragraphs

[0161.0.6.6] therefore, in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor length are at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 68-71 and 450-459 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.6] sees [0162.0.0.0] for disclosing of this paragraph

[0163.0.6.6] preferably, under stringent condition with Table I the 5th or 7 row, 68-71 and 450-459 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.6] sees [0164.0.0.0] for disclosing of this paragraph

[0165.0.6.6] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 68-71 and 450-459 capable shown in sequence).

Above [0166.0.0.6] and [0167.0.0.6] sees for disclosing of [0166.0.0.6] and [0167.0.0.6] these paragraphs [0166.0.0.0] and [0167.0.0.0]

[0168.0.6.6] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are with different as the contained sequence of sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable, but have kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the aminoacid sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding is with identical at least about 60% as the sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable, more preferably with identical at least about 70% as one of sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable, even more preferably with as the sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable at least about 80%, 90% or 95% homology, and most preferably with identical at least about 96%, 97%, 98% or 99% as the sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table II B the 7th row, 68-71 and 450-459 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 68-71 with 450-459 is capable, preferred Table II B the 7th row, 68-71 and the 450-459 aminoacid sequence shown in capable is at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 68-71 and 450-459 are capable, preferred Table II B the 7th row, 68-71 is identical at least about 60% with the sequence shown in 450-459 is capable, more preferably with as Table II the 5th or 7 be listed as, 68-71 and 450-459 are capable, preferred Table II B the 7th row, 68-71 is identical at least about 70% with one of sequence shown in 450-459 is capable, even more preferably with as Table II the 5th or 7 be listed as, 68-71 and 450-459 are capable, preferred Table II B the 7th row, sequence shown in 68-71 and 450-459 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 68-71 and 450-459 are capable, preferred Table II B the 7th row, sequence shown in 68-71 and 450-459 are capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.0] was to [0175.0.0.0] above [0169.0.0.6] to [0175.0.5.6] saw for disclosing of [0169.0.0.6] to [0175.0.5.6] these paragraphs

[0176.0.6.6] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in 68-71 and 450-459 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 68-71 and 450-459 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in 68-71 and 450-459 are capable has essentially identical character and discerns.

[0177.0.6.6] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the functional equivalent that nucleotide sequence obtained shown in 68-71 and 450-459 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 68-71 and 450-459 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in 68-71 and 450-459 are capable.

[0178.0.0.6] sees [0178.0.0.0] for disclosing of this paragraph

[0179.0.6.6] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I the 5th or 7 row, 68-71 and 450-459 capable shown in), and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding with as Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table II B the 7th is listed as, 68-71 and 450-459 capable shown in the homologous nucleic acid molecule of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to encoding sequence, in sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable, introduce sudden change.

[0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.6] to [0183.0.0.6] saw for disclosing of [0180.0.0.6] to [0183.0.0.6] these paragraphs

[0184.0.6.6] is employed to have as Table I the 5th or 7 row, 68-71 and 450-459 are capable, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in 68-71 and 450-459 are capable, perhaps come Table II the 5th or 7 row freely, 68-71 and 450-459 are capable, preferred Table II B the 7th row, the homologous compound of the nucleotide sequence of sequence shown in 68-71 and 450-459 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.6.6] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I the 5th or 7 row, 68-71 with 450-459 is capable, preferred Table I B the 7th row, 68-71 and the 450-459 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 68-71 with 450-459 is capable, other nucleotide sequence of not showing in preferred Table I B the 7th row, 68-71 and the 450-459 arbitrary sequence shown in capable.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 68-71 with 450-459 is capable, preferred Table I B the 7th row, 68-71 and the 450-459 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.6.6] also preferred the inventive method comprise as Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table II B the 7th row, 68-71 and 450-459 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table II B the 7th row, 68-71 and 450-459 capable shown in sequence identical.

[0187.0.6.6] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table II B the 7th row, 68-71 and 450-459 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table II B the 7th row, 68-71 and 450-459 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.6.6] still has the basic biologic activity of polypeptide of the present invention (being its active basic reduction that do not have) of giving fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active comparing with polypeptide expressed activity as Table II the 5th or 7 row, 68-71 and shown in 450-459 is capable and under the same conditions do not reduced substantially.In one embodiment, polypeptide of the present invention is to comprise as sequence shown in Table II B the 7th row, 68-71 and 450-459 are capable or by its homologue of forming.

[0189.0.6.6] as the homologous compound of sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or in (or even promotor of allos biology) it be replaced fully with active higher startup.Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.6], [0191.0.5.6], [00191.1.0.6] and [0192.0.0.6] to [0203.0.0.6] is for [0190.0.0.6], [0191.0.5.6], [0191.1.0.6] and [0192.0.0.6] is above disclosing of [0203.0.0.6] these paragraphs seen [0190.0.0.0], [0191.0.0.0], [0191.1.0.0] and [0192.0.0.0] is to [0203.0.0.0]

[0204.0.6.6] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table II B the 7th row, 68-71 and 450-459 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given fine chemicals quantity in biological or its part increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 7th row, 68-71 and 450-459 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as the primer shown in Table III the 7th row, 68-71 and 450-459 are capable or primer amplification from cDNA library or genomic library is obtained by using, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 68-71 and 450-459 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described peptide coding is as Table II the 5th or 7 row, 68-71 and 450-459 is capable, the structural domain of preferred Table II B the 7th row, 68-71 and the 450-459 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, nucleic acid molecule shown in 68-71 and 450-459 are capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 68-71 and 450-459 are capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A the 5th or 7 row, 68-71 and 450-459 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule do not form by Table I A or IB the 5th or 7 row, 68-71 and 450-459 by the sequence shown in capable.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 68-71 and 450-459 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as the peptide sequence shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as the protein of sequence shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable at least 30% identical and with Table II A or IIB the 5th or 7 row, 68-71 and 450-459 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.0] was to [0226.0.0.0] above [0205.0.0.6] to [0226.0.0.6] saw for disclosing of [0205.0.0.6] to [0226.0.0.6] these paragraphs

[0227.0.6.6] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 68-71 and the capable sequence or derivatives thereof of mentioning of 450-459, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid biosynthetic pathway of at least one lipid acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 68-71 and 450-459 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.0] was to [0230.0.0.0] above [0228.0.5.6] to [0230.0.5.6] saw for disclosing of [0228.0.5.6] to [0230.0.5.6] these paragraphs

[0231.0.6.6] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened the degraded alpha-linolenic acid simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.6] to [0276.0.0.6] saw for disclosing of [0232.0.0.6] to [0276.0.0.6] these paragraphs

[0277.0.5.6] sees [0277.0.5.5] for disclosing of this paragraph

[0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.6] to [0282.0.0.6] saw for disclosing of [0278.0.0.6] to [0282.0.0.6] these paragraphs

[0283.0.6.6] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-antibody as polypeptide shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or its antigen part, polypeptide (polypeptide for example of the present invention or its fragment) generation that it can comprise above-mentioned sequence or be made up of above-mentioned sequence by the standard technique utilization.Preferably specificity is in conjunction with the monoclonal antibody as polypeptide shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable.

[0284.0.0.6] sees [0284.0.0.0] for disclosing of this paragraph

[0285.0.6.6] the present invention relates to have as sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable in one embodiment.

[0286.0.6.6] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in 68-71 and 450-459 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in 68-71 and 450-459 are capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to and comprising more than a polypeptide as consensus sequence shown in Table IV the 7th row, 68-71 and 450-459 are capable.

[0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.6] to [0290.0.0.6] saw for disclosing of [0287.0.0.6] to [0290.0.0.6] these paragraphs

[0291.0.6.6] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise and contain plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in 68-71 and 450-459 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in 68-71 and 450-459 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable.

[0292.0.0.6] sees [0292.0.0.0] for disclosing of this paragraph

[0293.0.6.6] the present invention relates to give the polypeptide that each fine chemicals increases in one embodiment in biological or its part, described polypeptide is by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention coding.

In one embodiment, polypeptide of the present invention have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help to form as the sequence of nucleic acid molecule encoding shown in Table I A or IB the 5th or 7 row, 68-71 and 450-459 are capable.

[0294.0.6.6] in one embodiment, the present invention relates to have as Table II the 3rd row, the polypeptide of activity of proteins shown in 68-71 and 450-459 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in 68-71 and 450-459 are capable.

[0295.0.0.6], [0296.0.0.6] and [0297.0.5.6] are for [0295.0.0.6], and disclosing of [0296.0.0.6] and [0297.0.5.6] these paragraphs sees that top [0295.0.0.0] is to [0297.0.0.0]

The chemical of [00297.1.0.6] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 68-71 and 450-459 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.

[0298.0.6.6] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II the 5th or 7 row, 68-71 and 450-459, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable.

[0299.0.6.6] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of aminoacid sequence shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable.

[0300.0.6.6] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,68-71 and 450-459 capable shown in sequence different.Therefore, this polypeptide contains with complete amino acid sequence homology as sequence shown in Table II A or IIB the 5th or 7 row, 68-71 and 450-459 are capable and is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.6] sees [0301.0.0.0] for disclosing of this paragraph

The biologically-active moiety of [0302.0.6.6] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence of the aminoacid sequence shown in 68-71 and 450-459 are capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.6] sees [0303.0.0.0] for disclosing of this paragraph

[0304.0.6.6] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as the active of polypeptide shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.5.6], [0306.0.5.6] and [0306.1.0.6] are for [0305.0.5.6], and top [0305.0.0.0], [0306.0.0.0] and [0306.1.0.0] are seen in disclosing of [0306.0.5.6] and [0306.1.0.6] these paragraphs

Above [0307.0.0.6] and [0308.0.0.6] sees for disclosing of [0307.0.0.6] and [0308.0.0.6] these paragraphs [0307.0.0.0and[0308.0.0.0]

[0309.0.6.6] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned, as Table II the 5th or 7 row, be meant polypeptide shown in 68-71 and 450-459 are capable with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " non-polypeptide " during 68-71 and 450-459 are capable or " other polypeptide " are meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with the homology not basically of the polypeptide with protein active, for example do not give described in the literary composition active and from the protein of identical or different biology.

[0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.6] to [0334.0.0.6] saw for disclosing of [0310.0.0.6] to [0334.0.0.6] these paragraphs

[0335.0.6.6] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of one of sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.6] to [0342.0.0.6] saw for disclosing of [0336.0.0.6] to [0342.0.0.6] these paragraphs

[0343.0.6.6] as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 68-71 and 450-459 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as one of sequence shown in Table I the 5th or 7 row, 68-71 and 450-459 are capable or its homologue in a kind of organism.

[0344.0.0.6] is to [0350.0.0.6], [0351.0.5.6] and [0352.0.0.6] to [0361.0.0.6] for [0344.0.0.5] to [0350.0.0.5], [0351.0.5.5] and [0352.0.0.5] above disclosing of [0361.0.0.5] these paragraphs seen [0344.0.0.0] to [0361.0.0.0]

[0362.0.6.6] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or each fine chemicals of its part increases) carry out genetically modified any cell, the nucleic acid molecule of described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide (for example coding has the polypeptide of protein as polypeptide active shown in Table II the 3rd row, 68-71 and 450-459 are capable).Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, cytoactive improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Transgenosis with the active polypeptide of protein (as polypeptide shown in Table II the 3rd row, 68-71 and 450-459 are capable) is meant in the text because genomic regulation and control or manipulation, improves having protein (as Table II the 3rd row, 68-71 and polypeptide shown in 450-459 is capable) activity active or similar active polypeptide in cell or biological or its part.Example and the inventive method are described in above.

[0363.0.0.6], [0364.0.5.6] and [0365.0.0.6] to [0379.0.5.6] be for [0363.0.0.6], [0364.0.5.6] and [0365.0.0.6] above disclosing of [0379.0.5.6] these paragraphs seen [0363.0.0.0] to [0379.0.0.0]

[0380.0.5.6], [0381.0.0.6] and [0382.0.0.6] are for [0380.0.5.6], and top [0380.0.5.5], [0381.0.0.0] and [0382.0.0.0] are seen in disclosing of [0381.0.0.6] and [0382.0.0.6] these paragraphs

[0383.0.5.6], [0384.0.0.6], [0385.0.5.6] and [0386.0.5.6] is for [0383.0.5.6], [0384.0.0.6], top [0383.0.5.5] seen in disclosing of [0385.0.5.6] and [0386.0.5.6] these paragraphs, [0384.0.0.0], [0385.0.5.5] and [0386.0.5.5]

[0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.6] to [0392.0.0.6] saw for disclosing of [0387.0.0.6] to [0392.0.0.6] these paragraphs

[0393.0.6.6] the present invention relates to identify and gives the method that the compound that becomes more meticulous in the cell produces the gene product that increases that it comprises following step in one embodiment:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 68-71 and 450-459 is capable, preferred Table I B the 5th or 7 row, 68-71 and the 450-459 nucleic acid molecule shown in capable is hybridized under lax stringent condition nucleic acid molecule, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.0] was to [0416.0.0.0] above [0394.0.0.6] to [0415.0.0.6] and [0416.0.5.6] saw for disclosing of [0394.0.0.6] to [0415.0.0.6] and [0416.0.5.6] these paragraphs

[0417.0.5.6] and [0418.0.0.6] to [0430.0.0.6] for [0417.0.5.6] and [0418.0.0.6] above disclosing of [0430.0.0.6] these paragraphs seen [0417.0.5.5] and [0418.0.0.0] to [0430.0.0.0]

[0431.0.5.6], [0432.0.5.6], [0433.0.0.6] and [0434.0.0.6] be for [0431.0.5.6], and [0432.0.5.6], [0431.0.0.0] was to [0434.0.0.0] above the disclosing of [0433.0.0.6] and [0434.0.0.6] these paragraphs seen

[0435.0.5.5] was to [0440.0.5.5] above [0435.0.5.6] to [0440.0.5.6] saw for disclosing of [0435.0.5.6] to [0440.0.5.6] these paragraphs

Above [0441.0.0.6] and [0442.0.5.6] sees for disclosing of [0441.0.0.6] and [0442.0.5.6] these paragraphs [0441.0.0.0] and [0442.0.5.5]

[0443.0.0.6] sees [0443.0.0.0] for disclosing of this paragraph

Above [0444.0.5.6] and [0445.0.5.6] sees for disclosing of [0444.0.5.6] and [0445.0.5.6] these paragraphs [0444.0.5.5] and [0445.0.5.5]

[0446.0.0.0] was to [0453.0.0.0] above [0446.0.0.6] to [0453.0.0.6] saw for disclosing of [0446.0.0.6] to [0453.0.0.6] these paragraphs

Above [0454.0.5.6] and [0455.0.5.6] sees for disclosing of [0454.0.5.6] and [0455.0.5.6] these paragraphs [0454.0.5.5] and [0455.0.5.5]

[0456.0.0.6] sees [0456.0.0.0] for disclosing of this paragraph

[0457.0.5.5] was to [0460.0.5.5] above [0457.0.5.6] to [0460.0.5.6] saw for disclosing of [0457.0.5.6] to [0460.0.6.6] these paragraphs

[0461.0.6.6] embodiment 10: clone SEQ ID NO:5304 is used for expressing plant

[0462.0.0.6] sees [0462.0.0.0] for disclosing of this paragraph

[0463.0.6.6] passes through pcr amplification SEQ ID NO:5304 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.5.6], [0465.0.5.6] and [0466.0.0.6] are for [0464.0.5.6], and top [0464.0.5.5], [0465.0.5.5] and [0466.0.0.0] are seen in disclosing of [0465.0.5.6] and [0466.0.0.6] these paragraphs

[0467.0.6.6] selects following primer sequence for genes of SEQ ID NO:5304:

I) forward primer (SEQ ID NO:5640)

atggctatcg?acgaaaacaa?acag

Ii) reverse primer (SEQ ID NO:5641)

ttaaaaatct?tcgttagttt?ctgctac

[0468.0.0.0] was to [0479.0.0.0] above [0468.0.0.6] to [0479.0.0.6] saw for disclosing of [0468.0.0.6] to [0479.0.0.6] these paragraphs

[0480.0.6.6] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:5304

[0481.0.0.6] sees [0481.0.0.0] for disclosing of this paragraph

[0482.0.0.0] was to [0513.0.0.0] above [0482.0.0.6] to [0513.0.0.6] saw for disclosing of [0482.0.0.6] to [0513.0.0.6] these paragraphs

[0514.0.6.6] selects as another kind of, as people such as Geigenberger (Plant Cell﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect lipid acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??b2699	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.13	??1.32
??YER173W	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.15	??1.54
					??YGL205W	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.13	??1.24
??YIL150C	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??3.43	??3.43
					??b0050	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.11	??1.19
??b0251	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.14	??1.29
					??b0376	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.15	??1.34
??b0577	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.12	??1.96
					??b0849	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.13	??1.31

??ORF	Metabolite	Method	??Min	??Max
					??b2822	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.15	??1.20
??b3129	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.12	??1.26
					??b3457	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.15	??1.28
??b3462	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.13	??1.17
					??b3644	Alpha linolenic acid (C18:3 (c9, c12, c15))	??GC	??1.13	??1.36

[0515.0.5.6] the 2nd row have shown the lipid acid of being analyzed.The 4th row and the 5th row have shown the ratio of lipid acid between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

Above [0516.0.0.6] and [0517.0.5.6] sees for disclosing of [0516.0.0.6] and [0517.0.5.6] these paragraphs [0516.0.0.0] and [0517.0.0.0]

[0518.0.0.0] was to [0530.0.0.0] above [0518.0.0.6] to [0529.0.0.6] and [0530.0.5.6] saw for disclosing of [0518.0.0.6] to [0529.0.0.6] and [0530.0.5.6] these paragraphs

[0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.6] to [0530.6.0.6] saw for disclosing of [0530.1.0.6] to [0530.6.0.6] these paragraphs

[0531.0.0.0] was to [0534.0.0.0] above [0531.0.0.6] to [0533.0.0.6] and [0534.0.5.6] saw for disclosing of [0531.0.0.6] to [0533.0.0.6] and [0534.0.5.6] these paragraphs

[0535.0.0.0] was to [0538.0.0.0] above [0535.0.0.6] to [0537.0.0.6] and [0538.0.5.6] saw for disclosing of [0535.0.0.6] to [0537.0.0.6] and [0538.0.5.6] these paragraphs

[0539.0.0.0] was to [0543.0.0.0] above [0539.0.0.6] to [0542.0.0.6] and [0543.0.5.6] saw for disclosing of [0539.0.0.6] to [0542.0.0.6] and [0543.0.5.6] these paragraphs

[0544.0.0.0] was to [0552.0.0.0] above [0544.0.0.6] to [0547.0.0.6] and [0548.0.5.6] saw for disclosing of [0544.0.0.6] to [0547.0.0.6] and [0548.0.5.6] to [0552.0.0.6] these paragraphs to [0552.0.0.6]

[0552.1.0.6]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF title	Metabolite	??Min	??Max
				??YIL150C	Alpha linolenic acid (C18:3 (c9, c12, c15))	??1.469	??2.241

Table 2 shows that alpha-linolenic acid has increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YIL150c.

In one embodiment, yeast saccharomyces cerevisiae protein YIL150C or its homologue in maize plant, for example under the situation that the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue is enhanced, preferably, giving the fine chemicals alpha-linolenic acid is increased between 46% and 124%.

[0552.2.0.6] sees [0552.2.0.0] for disclosing of this paragraph

[0553.0.6.6]

1. produce the method for alpha-linolenic acid, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation alpha-linolenic acid.

2. produce the method for alpha-linolenic acid, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable, and described nucleic acid molecule gives that alpha-linolenic acid quantity increases in biology or its part;

B) contain just like Table I the 5th or 7 row, 68-71 and 450-459 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of alpha-linolenic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of alpha-linolenic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that alpha-linolenic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 68-71 and 450-459 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that alpha-linolenic acid quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that alpha-linolenic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 68-71 and 450-459 capable shown in consensus sequence polypeptide and give biology or its part in alpha-linolenic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give alpha-linolenic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded alpha-linolenic acid.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded alpha-linolenic acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 68-71 and 450-459 are capable, and described nucleic acid molecule gives that alpha-linolenic acid quantity increases in biology or its part;

B) contain just like Table I the 5th or 7 row, 68-71 and 450-459 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of alpha-linolenic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of alpha-linolenic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that alpha-linolenic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 68-71 and 450-459 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that alpha-linolenic acid quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that alpha-linolenic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 68-71 and 450-459 capable shown in consensus sequence polypeptide and give biology or its part in alpha-linolenic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give alpha-linolenic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A the 5th or 7 row, 68-71 and 450-459 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 68-71 and 450-459 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in alpha-linolenic acid quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part alpha-linolenic acid quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps alpha-linolenic acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the alpha-linolenic acid level that will measure or expression of polypeptides level and standard alpha-linolenic acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the alpha-linolenic acid generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of alpha-linolenic acid quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of alpha-linolenic acid quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the alpha-linolenic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that alpha-linolenic acid increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce alpha-linolenic acid;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) the alpha-linolenic acid level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give alpha-linolenic acid level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the alpha-linolenic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that alpha-linolenic acid quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce alpha-linolenic acid;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) the alpha-linolenic acid level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give alpha-linolenic acid level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that alpha-linolenic acid increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control alpha-linolenic acid level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make the anti-alpha-linolenic acid synthetic weedicide that suppresses of plant.

[0554.0.0.6] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.7] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.7] sees [0001.0.0.0] for disclosing of this paragraph.

[0002.0.7.7] lipid acid and triacylglycerol have multiple application at food and fodder industry, cosmetic industry and medicine department.Depend on that they are saturated or unsaturated fatty acids of free or the triacylglycerol with saturated or unsaturated fatty acid content of raising, they are applicable to multiple application; Therefore, for example, polyunsaturated fatty acid (=PUFA) can add in the infant food to improve nutritive value.Multiple lipid acid and triacylglycerol mainly be obtained from microorganism such as fungi or oil-produced vegetable, comprise plant plankton and algae, for example soybean, rape, Sunflower Receptacle and other, the form of the normally triacylglycerol that obtains therein.

[0003.0.7.7] stearic acid (=octadecanoic acid) is one of many useful saturated fatty acid types, and it is from many animal and plant fat and oils.It is the waxy solid of fusing about 70 ℃.Usually, stearic acid is by begin preparation with water treatment animal tallow preparation or the hydrogenization by described oils from plant oil under high pressure and high temperature.It can be used for making the composition of candle, soap and makeup or is used for softened rubber.

[0004.0.7.7] microorganism such as genus mortierella or oil-produced vegetable such as soybean, Semen Brassicae campestris or Sunflower Receptacle or algae such as Crytocodinium belong to or the brown algae genus is the main common source of containing the oils of lipid acid, the form of the normally triacylglycerol that obtains therein.Alternatively, they can advantageously obtain from animal such as fish.Free fatty acids is advantageously by using highly basic (as potassium hydroxide or sodium hydroxide) hydrolysis preparation.

[0005.0.5.7] sees [0005.0.5.5] for disclosing of this paragraph

[0006.0.7.7] is different with most of saturated fattys, as if stearic acid can not improve the cholesterol levels in the blood, because the liver enzyme converts it into unsaturated fatty acids in digestive process.

[0007.0.7.7] stearic acid is one of modal longer chain fatty acid.At many foods, for example found stearic acid in tallow and the theobroma oil.As mentioned above, it is used as lubricant widely and is used as tenderizer in rubber in soap, makeup, food product pack, deodorant stick, toothpaste.

[0008.0.7.7] is inspirer to be studies show that, as the stearic acid of one of existing fatty ingredient in the chocolate theobroma oil thrombocyte is had some positive influences.Think that its mechanism is, stearic acid is potential activated blood platelet by participating in arachidonic acid metabolism (thromboxane A2 and the effective anti-freezing collection compound prostacyclin I2 that comprise effective aggegation compound).

[0009.0.7.7] as mentioned above, lipid acid can be used for multiple different purposes, for example is used for makeup, medicine and feed and food.

Therefore [0010.0.7.7] improves the productivity of this kind lipid acid and improves food and the quality of animal-feed is different industrial vital tasks.

[0011.0.7.7] in order to ensure the high productivity of certain lipid acid in plant or the microorganism, is necessary to operate transformation to the natural biological of lipid acid in the described biology is synthetic.

[0012.0.7.7] therefore still exists great demand new and more suitable gene, and described genes encoding participates in fatty acid biological synthetic enzyme and can produce some lipid acid and not form unnecessary byproduct in the particular industry scale.Selecting to be used for biosynthetic gene, above-mentioned two features are particularly importants.On the one hand, but still need to obtain the development of the highest intrinsic energy lipid acid, on the other hand, produce byproduct in process of production as few as possible.

[0013.0.0.7] sees [0013.0.0.0] for disclosing of this paragraph

[0014.0.7.7] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is stearic acid or contains stearic triglyceride level, lipid, oils or fat.Therefore, in the present invention, term " fine chemicals " is meant " stearic acid and/or contain stearic triglyceride level, lipid, oils or fat " as used herein.In addition, term " fine chemicals " also refers to comprise stearic acid and/or contains stearic triglyceride level, lipid, oils or fatty fine chemicals as used herein.

[0015.0.7.7] an embodiment, term " fine chemicals " meaning is meant stearic acid and/or contains stearic triglyceride level, lipid, oils or fat.In whole specification sheets, term " fine chemicals " meaning is meant stearic acid and/or contains stearic triglyceride level, lipid, oils or fat, stearic acid or its salt, ester, thioesters or free form or in conjunction with the stearic acid of other compound such as triacylglycerol, glycolipid, phosphatide etc.In preferred embodiments, term " fine chemicals " meaning is meant free stearic acid or its salt or is bonded to the stearic acid of triacylglycerol.Triacylglycerol, lipid, oils, fat or its lipid mixtures should refer to any triacylglycerol, lipid, oils and/or contain any combination or free stearic fat, for example sphingolipid, phosphoglyceride, lipid, glycolipid class such as sphingoglycolipid, phosphatide such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, glycerine Serine, phosphatidylinositols or two glycerine Serines or monoacylglycerol, diacylglycerol or triacylglycerol or other fatty acid ester such as acetyl-CoA thioesters, it contains other saturated or unsaturated fatty acids in fatty acid molecule.

In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning be meant to have the active at least a chemical compound of above-mentioned fine chemicals.

[0016.0.7.7] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce b2699, b2095, b3256, b2699, b1093, YOR024W, YBR089C, YFR042W, YIL150C, YDR513W, YLR010C, b0161, b1896 and/or b3457 protein or have Table I the 5th or 7 row, 72-81 and 460-462 capable shown in the activity of proteins of the coded peptide sequence of nucleic acid molecule; With

(b) producing fine chemicals in allowing described biology is to make biological growth under stearic acid or the condition that comprises stearic fine chemicals.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II the 3rd row, 72-81 and 460-462 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, 72-81 and 460-462 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) allowing the generation fine chemicals, particularly making biological growth under the stearic condition.

Above [0017.0.0.7] and [0018.0.0.7] sees for disclosing of [0017.0.0.7] and [0018.0.0.7] these paragraphs [0017.0.0.0] and [0018.0.0.0]

The method that [0019.0.7.7] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare the fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 72-81 and 460-462 are capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 72-81 and 460-462 were capable.

[0020.0.7.7] be surprisingly found out that, at least a as the yeast saccharomyces cerevisiae protein shown in Table II the 3rd row, 75-80 are capable and/or at least a such as Table II the 3rd row, 72-74,81 and e. coli k12 protein in Arabidopis thaliana the transgene expression of 460-462 shown in capable give the raising of conversion plant Threonine (or fine chemicals) content.

[0021.0.0.7] sees [0021.0.0.0] for disclosing of this paragraph

The sequence of [0022.0.7.7] e. coli k12 b3256 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be acetyl CoA carboxylase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary acetyl-CoA carboxylase or its homologue as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the activity of acetyl-CoA carboxylase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary acetyl-CoA carboxylase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the auxilliary proteolytic enzyme of DNA dependency ATP enzyme or DNA and ATP dependency.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary DNA dependency ATP enzyme or DNA and the auxilliary proteolytic enzyme of ATP dependency or its homologue as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the auxilliary protease activities of DNA dependency ATP enzyme or DNA and ATP dependency is enhanced or produces in the methods of the invention, and for example the activity from colibacillary DNA dependency ATP enzyme or DNA and the auxilliary proteolytic enzyme of ATP dependency or its homologue is enhanced or produces.

The sequence of e. coli k12 b2095 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition tagatose-6-phosphokinase 1.Therefore, in one embodiment, the inventive method comprises the purposes of the tagatose from colibacillary supposition as shown here-6-phosphokinase 1 or its homologue, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the activity of Jia Ding tagatose-6-phosphokinase 1 is enhanced or produces in the methods of the invention, for example is enhanced or produces from the tagatose-6-phosphokinase 1 of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b1093 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 3-oxo acyl group-[acyl group-carrier-protein] reductase enzyme.Therefore; in one embodiment; the inventive method comprises the purposes from colibacillary 3-oxo acyl group-[acyl group-carrier-protein] reductase enzyme or its homologue as shown here; it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment; the activity of 3-oxo acyl group-[acyl group-carrier-protein] reductase enzyme is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 3-oxo acyl group-[acyl group-carrier-protein] reductase enzyme or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YOR024W is committed to the EMBL protein sequence database by de Haan in July, 1996, and its cytoactive is not still characterized.It may be a membranin.Therefore, in one embodiment, the inventive method comprises the purposes of YOR024W or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the activity of YOR024W is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YOR024W of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YBR089C-A has been published in Feldmann etc., EMBO J., and 13 (24), 5795-5809 (1994) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.It shows with Mammals high mobility group protein 1 and 2 to have homology.Its function may be unnecessary to height homologous gene NHP6A.In addition, it shows and the nonhistones chromatin protein N hp6bp homology of high speed swimming family.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YBR089C-A from yeast saccharomyces cerevisiae as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the YBR089C-A activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YBR089C-A protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YFR042W has been published in Murakami etc., Nat.Genet.10 (3), and 261-268 (1995) and Goffeau etc., Science 274 (5287), 546-547,1996, and the still clear sign of its cytoactive.It is " the necessary protein of cell survival " seemingly.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YFR042W from yeast saccharomyces cerevisiae as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the YFR042W activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YFR042W protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Churcher etc., Nature 387 (6632 supplementary issue), and 84-87 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.It is " S phase (DNA synthetic) initial or finish necessary protein " seemingly.Therefore, in one embodiment, the inventive method comprises the purposes of YIL150C or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the YIL150C activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YIL150C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YDR513w has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be described to be glutaredoxin (thiol transferase, glutathione reductase).Therefore, in one embodiment, the inventive method comprises the purposes of YDR513w or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the activity of glutaredoxin is enhanced or produces in the methods of the invention, for example is enhanced or produces from the glutaredoxin of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YLR010c has been published in Johnston etc., Nature 387 (6632 supplementary issue), and 87-90 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.It seemingly " participates in the protein of the telomere approach relevant with Stn1 ".Therefore, in one embodiment, the inventive method comprises the purposes of YLR010C or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the YLR010C activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YLR010C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0161 (accession number NP_414703) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary pericentral siphon serine protease or its homologue as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the activity of pericentral siphon serine protease is enhanced or produces in the methods of the invention, and for example the activity from colibacillary pericentral siphon serine protease or its homologue is enhanced or produces.

The sequence of e. coli k12 b1896 (accession number NP_416410) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be trehalose-6-phosphate synthase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary trehalose-6-phosphate synthase or its homologue as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the activity of trehalose-6-phosphate synthase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary trehalose-6-phosphate synthase or its homologue is enhanced or produces.

The sequence of e. coli k12 b3457 (accession number NP_417914) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary high-affinity branched-chain amino acid translocator or its homologue as shown here, it is used for biological or its part produces fine chemicals, be stearic acid and/or contain stearic triglyceride level, lipid, oils and/or fat as described, particularly increases stearic acid and/or contains stearic triglyceride level, lipid, oils and/or fat, the preferred stearic quantity of free or combining form.In one embodiment, the activity of high-affinity branched-chain amino acid translocator is enhanced or produces in the methods of the invention, and for example the activity from colibacillary high-affinity branched-chain amino acid translocator or its homologue is enhanced or produces.

The homologous compound (=homologue) of [0023.0.7.7] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.

Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with protein shown at least a Table II the 3rd row, 72-81 and 460-462 are capable, for example have by contain Table I the 5th or 7 row, 72-81 and 460-462 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in Table II 75-80 is capable in the polypeptide homologue of any one be to have a same or similar active homologue, particularly active increasing gives that fine chemicals content increases in the biology, and described homologue is from eukaryote.In one embodiment, Table II the 3rd row, the 63rd, 64 and 436-449 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 75-80 are capable is to have a same or similar active homologue, and particularly active increasing gives that fine chemicals content increases in biology or its part, and described homologue is from fungi.In one embodiment, Table II the 3rd row, 72-74,81 and 460-462 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Proteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 75-80 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Ascomycota.In one embodiment, Table II the 3rd row, 72-74,81 and 460-462 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 75-80 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from yeast.In one embodiment, Table II the 3rd row, 72-74,81 and 460-462 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the enterobacteria order.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 75-80 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.In one embodiment, Table II the 3rd row, 72-74,81 and 460-462 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from enterobacteriaceae.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 75-80 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetes.In one embodiment, Table II the 3rd row, 72-74,81 and 460-462 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Escherichia.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 75-80 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetaceae.In one embodiment, the homologue of polypeptide was to have same or similar active homologue shown in Table II the 3rd row, 75-80 were capable, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.

Above [0023.1.0.7] and [0024.0.0.7] sees for disclosing of [0023.1.0.7] and [0024.0.0.7] these paragraphs [0023.1.0.0] and [0024.0.0.0]

[0025.0.7.7] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause in biology or its part, the preferred described biomass cells stearic acid and/or contain that stearic triglyceride level, lipid, oils or fat level increase and protein has as proteinic above-mentioned activity shown in Table II the 3rd row, 72-81 and 460-462 are capable, then described protein or polypeptide have " as activity of proteins shown in Table II the 3rd row, 72-81 and 460-462 are capable ".In this manual, if this kind protein or polypeptide still have Table II the 3rd row, proteinic biology or the enzyme activity shown in 72-81 and 460-462 are capable, if promptly with Table II the 3rd row, yeast saccharomyces cerevisiae protein shown in 75-80 is capable is compared and/or is listed as with Table II the 3rd, 72-74,81 compare with the protein of e. coli k12 shown in the 460-462, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

Above [0025.1.0.7] and [0025.2.0.7] sees for disclosing of [0025.1.0.7] and [0025.2.0.7] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.7] to [0033.0.0.7] saw for disclosing of [0026.0.0.7] to [0033.0.0.7] these paragraphs

[0034.0.7.7] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 72-81 and protein shown in 460-462 is capable or by the active protein expression level of the protein of Table I the 5th row, 72-81 and nucleic acid molecule encoding shown in 460-462 is capable or its homologue (as Table I the 7th row, 72-81 and homologue shown in 460-462 is capable) or active aspect different, and it is difference aspect biological chemistry or genetics reason, and therefore shows the fine chemicals quantity that increases.

[0035.0.0.0] was to [0039.0.0.0] above [0035.0.0.7] to [0038.0.0.7] and [0039.0.5.7] saw for disclosing of [0035.0.0.7] to [0038.0.0.7] and [0039.0.5.7] these paragraphs

Above [0040.0.0.7] to [0044.0.0.7] sees for disclosing of [0040.0.0.7] to [0044.0.0.7] these paragraphs [0035.0.0.0] and [0044.0.0.0]

[0045.0.7.7] in one embodiment, at e. coli k12 protein b3256 or its homologue, for example under the activity situation about being enhanced of acetyl CoA carboxylase protein (for example shown in Table II the 5th or 7 row, the 72nd row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 17% and 25% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example under the situation that the activity of the auxilliary protease protein matter of DNA dependency ATP enzyme or DNA and ATP dependency (for example shown in Table II the 5th or 7 row, the 73rd row) is enhanced, preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 22% and 83% or more between.

At e. coli k12 protein b2095 or its homologue, for example under the activity situation about being enhanced of Jia Ding tagatose-6-phosphokinase 1 (for example shown in Table II the 5th or 7 row, the 74th row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 17% and 26% or more between.

In one embodiment; at e. coli k12 protein b1093 or its homologue; for example under the activity situation about being enhanced of 3-oxo acyl group-[acyl group-carrier-protein] reductase enzyme (for example shown in Table II the 5th or 7 row, the 81st row); preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 16% and 31% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR024W or its homologue, for example under the activity situation about being enhanced of " not profiling protein matter YOR024W " (it is " possible membranin " seemingly) (for example shown in Table II the 5th or 7 row, the 75th row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 16% and 40% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, under the situation that as if activity of for example " not profiling protein matter YBR089C-A " (its " having homology with Mammals high mobility group protein 1 and 2 ") (for example shown in Table II the 5th or 7 row, the 76th row) be enhanced, preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 36% and 134% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFR042W or its homologue, under the activity situation about being enhanced of for example " not profiling protein matter YFR042W " (it is " necessary protein of cell survival " seemingly) (for example shown in Table II the 5th or 7 row, the 77th row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 19% and 26% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, under the situation that the activity of for example " not profiling protein matter YIL150C " (it may be " S phase (DNA synthetic) initial or finish necessary protein ") (for example shown in Table II the 5th or 7 row, the 78th row) is enhanced, preferably, in one embodiment, give fine chemicals, preferred stearic acid be increased in 34% and 220% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR513W or its homologue, under the activity situation about being enhanced of for example " glutaredoxin (thiol transferase; glutathione reductase) " (for example shown in Table II the 5th or 7 row, the 79th row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 16% and 51% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YLR010C or its homologue, under the activity situation about being enhanced of for example " not profiling protein matter YLR010C " (it may be " participating in the protein of the telomere approach relevant with Stn1 ") (for example shown in Table II the 5th or 7 row, the 80th row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 16% and 76% or more between.

In one embodiment, at e. coli k12 protein b0161 or its homologue, for example under the activity situation about being enhanced of pericentral siphon serine protease (for example shown in Table II the 5th or 7 row, the 460th row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 16% and 129% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the activity situation about being enhanced of trehalose-6-phosphate synthase (for example shown in Table II the 5th or 7 row, the 461st row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 23% and 30% or more between.

In one embodiment, at e. coli k12 protein b3457 or its homologue, for example under the activity situation about being enhanced of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) (for example shown in Table II the 5th or 7 row, the 462nd row), preferably, give in one embodiment fine chemicals, preferred stearic acid be increased in 18% and 34% or more between.

[0046.0.7.7] is at e. coli k12 protein b3256 or its homologue, for example under the situation that the acetyl-CoA carboxylase activity of proteins is enhanced, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increase.

At e. coli k12 protein b2699 or its homologue, for example under the situation that the activity of the auxilliary protease protein matter of DNA dependency ATP enzyme or DNA and ATP dependency is enhanced, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increase.

At e. coli k12 protein b2095 or its homologue, for example under the situation that the activity of Jia Ding tagatose-6-phosphokinase 1 is enhanced, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increase.

At e. coli k12 protein b1093 or its homologue; for example under the situation that the activity of 3-oxo acyl group-[acyl group-carrier-protein] reductase enzyme is enhanced; preferably, give in one embodiment fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

At yeast saccharomyces cerevisiae protein YOR024W or its homologue, for example under the situation that the activity of " not profiling protein matter YOR024W " (it is " possible membranin " seemingly) is enhanced, preferably, give in one embodiment fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

At yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, under as if the situation that the activity of for example " not profiling protein matter YBR089C-A " (it is " having homology with Mammals high mobility group protein 1 and 2 ") is enhanced, preferably, give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increase.

At yeast saccharomyces cerevisiae protein YFR042W or its homologue, under the situation that the activity of for example " not profiling protein matter YFR042W " (it is " the necessary protein of cell survival " seemingly) is enhanced, preferably, in one embodiment, give fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

At yeast saccharomyces cerevisiae protein YIL150C or its homologue, under the situation that the activity of for example " not profiling protein matter YIL150C " (it may be " S phase (DNA synthetic) initial or finish necessary protein ") is enhanced, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat and increase.

At yeast saccharomyces cerevisiae protein YDR513W or its homologue, for example under the situation that the activity of " glutaredoxin (thiol transferase; glutathione reductase) " is enhanced, preferably, in one embodiment, give fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

At yeast saccharomyces cerevisiae protein YLR010C or its homologue, under the situation that the activity of for example " not profiling protein matter YLR010C " (it may be " participating in the protein of the telomere approach relevant with Stn1 ") is enhanced, preferably, in one embodiment, give fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

At e. coli k12 protein b0161 or its homologue, for example under the situation that the activity of pericentral siphon serine protease is enhanced, preferably, in one embodiment, give fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that the activity of trehalose-6-phosphate synthase is enhanced, preferably, in one embodiment, give fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

In one embodiment, at e. coli k12 protein b3457 or its homologue, for example under the situation that the activity of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) is enhanced, preferably, in one embodiment, give fine chemicals, preferably give fine chemicals and contain stearic triglyceride level, lipid, oils and/or fat increases.

Above [0047.0.0.7] to [0048.0.0.7] sees for disclosing of [0047.0.0.7] and [0048.0.0.7] these paragraphs [0047.0.0.0] and [0048.0.0.0]

[0049.0.7.7] has to give and improves each fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, the polypeptide of consensus sequence shown in 72-81 and 460-462 are capable, perhaps as Table II the 5th and 7 row, its function homologue described in polypeptide or the literary composition shown in the capable disclosed aminoacid sequence of 72-81 and 460-462, perhaps (for example be listed as by Table I the 5th and 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 72-81 and 460-462 are capable or its function homologue as herein described) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.7.7] for the purposes of the present invention, term " stearic acid " also comprises corresponding salt, for example stearic sylvite or sodium salt or have amine such as the stearic salt of diethylamine.

Above [0051.0.5.7] and [0052.0.0.7] sees for disclosing of [0051.0.5.7] and [0052.0.0.7] these paragraphs [0051.0.0.0] and [0052.0.0.0]

[0053.0.7.7] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having as the active polypeptide of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 7th row, 72-81 and 460-462 capable shown in)) and is expressed and increase, and has the stearic activity of the raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example having as the active polypeptide of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 7th row, 72-81 and 460-462 capable shown in)) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of the raising stearic acid described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give the raising stearic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having) as the active polypeptide of protein or its homologue shown in Table II the 3rd row, 72-81 and 460-462 are capable (for example as Table II the 7th row, 72-81 and 460-462 are capable shown in), perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give the raising stearic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 7th row, 72-81 and 460-462 capable shown in);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give the raising stearic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 7th row, 72-81 and 460-462 capable shown in);

(f) express the transgenosis of coded protein, described protein give the raising stearic acid that has described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 7th row, 72-81 and 460-462 capable shown in);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have the raising stearic acid described in the literary composition active, by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 7th row, 72-81 and 460-462 capable shown in);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide or the used polypeptide of the inventive method (for example having as the active polypeptide of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 7th row, 72-81 and 460-462 capable shown in)).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.7.7] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example after active expression of polypeptides of protein shown in Table II the 3rd row, 72-81 and 460-462 are capable or its homologue (for example Table II the 5th or 7 row, 72-81 and 460-462 capable shown in polypeptide) or activity, give stearic acid and increase improving coded protein or have.

[0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.7] to [0067.0.0.7] saw for disclosing of [0055.0.0.7] to [0067.0.0.7] these paragraphs

Above [0068.0.5.7] and [0069.0.5.7] sees for disclosing of [0068.0.5.7] and [0069.0.5.7] these paragraphs [0068.0.0.0] and [0069.0.0.0]

Above [0070.0.6.7] and [0071.0.5.7] sees for disclosing of [0070.0.6.7] and [0071.0.5.7] these paragraphs [0070.0.5.5] and [0071.0.0.0]

[0072.0.7.7] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds except stearic acid, contain stearic triacylglycerol, lipid, oils and/or the fat and to also have other compound, for example palmitinic acid, Zoomeric acid, stearic acid, oleic acid, alpha-linolenic acid and/or linolic acid.

[0073.0.7.7] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of protein with polypeptide of the present invention or the used polypeptide active of the inventive method or its homologue (for example Table II the 5th or 7 row, 72-81 and 460-462 capable shown in) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of promptly giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and other optional free and/or conjugated fatty acid.

[0074.0.5.7] sees [0074.0.5.5] for disclosing of this paragraph

[0075.0.0.0] was to [0084.0.0.0] above [0075.0.0.7] to [0084.0.0.7] saw for disclosing of [0075.0.0.7] to [0084.0.0.7] these paragraphs

[0085.0.7.7] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I the 5th and 7 row, 72-81 and 460-462 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as Table I the 5th and 7 row, 72-81 and nucleotide sequence or derivatives thereof shown in 460-462 is capable, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

Above [0086.0.0.7] and [0087.0.0.7] sees for disclosing of [0086.0.0.7] and [0087.0.0.7] these paragraphs [0086.0.0.0] and [0087.0.0.0]

[0088.0.7.7] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified fatty acid content.Because the nutritive value that for example is used for the plant of poultry depends on above-mentioned indispensable fatty acid and as the big scale of construction of the lipid acid in fodder energy source, so this is very important for the plant breeder.Improve or produced as Table II the 5th or 7 row, 72-81 and 460-462 capable shown in after the activity of polypeptide, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, thus the transgenic plant that produced grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0088.1.0.7], [0089.0.0.7], [0090.0.0.7] and [0091.0.5.7] is for [0088.1.0.7], [0089.0.0.7], top [0088.1.0.0] seen in disclosing of [0090.0.0.7] and [0091.0.5.7] these paragraphs, [0089.0.0.0], [0090.0.0.0] and [0091.0.0.0]

[0092.0.0.0] was to [0094.0.0.0] above [0092.0.0.7] to [0094.0.0.7] saw for disclosing of [0092.0.0.7] to [0094.0.0.7] these paragraphs

[0095.0.5.7], [0096.0.5.7] and [0097.0.5.7] are for [0095.0.5.7], and top [0095.0.5.5], [0096.0.5.5] and [0097.0.0.0] are seen in disclosing of [0096.0.5.7] and [0097.0.5.7] these paragraphs

[0098.0.7.7] in preferred embodiments, fine chemicals (stearic acid) be produce according to the present invention and carry out isolating where necessary.It is favourable as palmitinic acid, stearic acid, Zoomeric acid, oleic acid and/or linolic acid mixture or other fatty acid mixt that the method according to this invention produces other lipid acid.

Above [0099.0.5.7] and [0100.0.5.7] sees for disclosing of [0099.0.5.7] and [0100.0.5.7] these paragraphs [0099.0.5.5] and [0100.0.5.5]

Above [0101.0.5.7] and [0102.0.5.7] sees for disclosing of [0101.0.5.7] and [0102.0.5.7] these paragraphs [0101.0.0.0] and [0102.0.5.5]

[0103.0.7.7] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as the sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as the sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer as sequence shown in Table III the 7th row, 72-81 and 460-462 are capable obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as the consensus sequence of sequence shown in Table IV the 7th row, 72-81 and 460-462 are capable and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, 72-81 and 460-462 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

Above [0103.1.0.7] and [0103.2.0.7] sees for disclosing of [0103.1.0.7] and [0103.2.0.7] these paragraphs [0103.1.0.0] and [0103.2.0.0]

[0104.0.7.7] in one embodiment, sequence had one or more Nucleotide different shown in sequence, preferred Table I A the 5th or 7 row, 72-81 and 460-462 shown in the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 72-81 and 460-462 are capable were capable, and not by Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I A the 5th or 7 row, 72-81 and 460-462 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 72-81 and 460-462 capable shown in sequence, preferred Table I A the 5th or 7 row, 72-81 and 460-462 capable shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 72-81 and 460-462 capable shown in sequence, preferred Table I A the 5th or 7 row, 72-81 and 460-462 capable shown in polypeptide of sequence.

Above [0105.0.0.7] to [0107.0.0.7] sees for disclosing of [0105.0.0.7] to [0107.0.0.7] these paragraphs [0105.0.0.0] and [0107.0.0.0]

Advantageously improved in [0108.0.7.7] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in 72-81 and 460-462 are capable, from Table II the 5th or 7 row, aminoacid sequence shown in 72-81 and 460-462 are capable is derived or is listed as from containing Table IV the 7th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 72-81 and 460-462 are capable, perhaps its coding has as Table II the 5th or 7 row, protein enzymic activity shown in 72-81 and 460-462 are capable or bioactive polypeptide or for example give derivative or the homologue that the fine chemicals linolic acid increases behind its expression or active the increasing.

[0109.0.5.7] sees [0109.0.0.0] for disclosing of this paragraph

[0110.0.7.7] helps the nucleic acid molecule of the polypeptide that the inventive method and coding have the used polypeptide active of the used or of the present invention process of the inventive method (for example as protein shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or by as the protein of nucleic acid molecule encoding shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable or its homologue (shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable)) and can determine from generally open database.

[0111.0.0.7] sees [0111.0.0.0] for disclosing of this paragraph

The nucleic acid molecule that uses in [0112.0.7.7] the inventive method is the isolated nucleic acid sequences form, its coding has as the polypeptide of polypeptide active shown in Table II the 3rd row, 72-81 and 460-462 are capable or has polypeptide as peptide sequence shown in Table II the 5th and 7 row, 72-81 and 460-462 are capable, and gives each fine chemicals and increase.

Above [0113.0.0.7] to [0120.0.0.7] sees for disclosing of [0113.0.0.7] to [0120.0.0.7] these paragraphs [0113.0.0.0] and [0120.0.0.0]

[0121.0.7.7] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with the difference that one or more amino acid moleculars are arranged as peptide sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or its functional homologue as herein described, described artificial sequence is preferably given aforementioned activity, and promptly giving each fine chemicals after improving its activity increases.

Above [0122.0.0.7] to [0127.0.0.7] sees for disclosing of [0122.0.0.7] to [0127.0.0.7] these paragraphs [0122.0.0.0] and [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.7.7] polymerase chain reaction (PCR) amplification is used (for example as the primer shown in Table III the 7th row, 72-81 and 460-462 are capable) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 72-81 and 460-462 capable shown in sequence or produce as sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable.

[0129.0.7.7] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (peptide sequence particularly of the present invention).Point out in the comparison shown in the figure of the conservative region of polypeptide of the present invention.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence was from described comparison shown in Table IV the 7th row, 72-81 and 460-462 were capable.

[0130.0.7.7] sees [0130.0.0.0] for disclosing of this paragraph.

[0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.7] to [0138.0.0.7] saw for disclosing of [0131.0.0.7] to [0138.0.0.7] these paragraphs

[0139.0.7.7] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 5th or 7 row, 72-81 and 460-462 capable shown in sequence hybridization, and coding is expressed and is had the active peptide of the stearic acid of increasing.

Above [0140.0.0.7] to [0146.0.0.7] sees for disclosing of [0140.0.0.7] to [0146.0.0.7] these paragraphs [0140.0.0.0] and [0146.0.0.0]

[0147.0.7.7] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 5th or 7 row, 72-81 and 460-462 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.7.7] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 72-81 and 460-462 are capable, preferred Table I B the 5th or 7 row, nucleotide sequence shown in 72-81 and 460-462 are capable or its funtion part homology are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases fine chemicals.

[0149.0.7.7] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 5th or 7 row, 72-81 and 460-462 capable shown in one of nucleotide sequence or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (increasing as giving each fine chemicals) and as Table II the 5th or 7 row, 72-81 with 460-462 is capable, preferred Table II B the 5th or 7 row, 72-81 and the 460-462 protein shown in capable.

[00149.1.0.7] randomly, with Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 5th or 7 row, 72-81 and 460-462 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity is for being known activity or being used for these protein of note as the protein shown in Table II the 3rd row, 72-81 and 460-462 are capable.

[0150.0.7.7] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 5th or 7 row, 72-81 and 460-462 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving methionine(Met) when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 72-81 and 460-462 capable shown in sense strand, Table I the 5th or 7 row, 72-81 and the 460-462 of one of sequence capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 72-81 and 460-462 capable shown in the right PCR of primer will produce as Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 7th is listed as, 72-81 and 460-462 capable shown in the fragment of polynucleotide sequence.

[0151.0.0.7] sees [0151.0.0.0] for disclosing of this paragraph

[0152.0.7.7] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as the abundant homology of aminoacid sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable, make this albumen or its part keep to participate in produce the ability of fine chemicals, specifically be included in plant or the microorganism as described above or the stearic activity of raising as be shown in the examples.

[0153.0.7.7] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as protein shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or its part have Table II the 5th for example as herein described or 7 row, 72-81 and 460-462 capable shown in the activity of polypeptide.

[0154.0.7.7] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

Above [0155.0.0.7] and [0156.0.0.7] sees for disclosing of [0155.0.0.7] and [0156.0.0.7] these paragraphs [0155.0.0.0] and [0156.0.0.0]

[0157.0.7.7] the present invention relate in addition owing to the genetic code degeneracy be different from Table I the 5th or 7 row, 72-81 and 460-462 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase) for example comprises as the polypeptide of consensus sequence shown in Table IV the 7th row, 72-81 and 460-462 are capable or as the nucleic acid molecule of polypeptide shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or its function homologue.Preferably, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as the consensus sequence shown in Table IV the 7th row, 72-81 and 460-462 are capable or as polypeptide or its function homologue shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as the consensus sequence shown in Table IV the 7th row, 72-81 and 460-462 are capable or as the basic homology of aminoacid sequence of polypeptide shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I A the 5th or 7 row, 72-81 and the 460-462 sequence shown in capable.Preferably, nucleic acid molecule of the present invention is a functional homologous compound or identical with nucleic acid molecule shown in Table I B the 5th or 7 row, 72-81 and 460-462 are capable.

[0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.7] to [0160.0.0.7] saw for disclosing of [0158.0.0.7] to [0160.0.0.7] these paragraphs

[0161.0.7.7] therefore, in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor length are at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 72-81 and 460-462 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.7] sees [0162.0.0.0] for disclosing of this paragraph

[0163.0.7.7] preferably, under stringent condition with Table I the 5th or 7 row, 72-81 and 460-462 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.7] sees [0164.0.0.0] for disclosing of this paragraph

[0165.0.7.7] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 72-81 and 460-462 capable shown in sequence).

Above [0166.0.0.7] and [0167.0.0.7] sees for disclosing of [0166.0.0.7] and [0167.0.0.7] these paragraphs [0166.0.0.0] and [0167.0.0.0]

[0168.0.7.7] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are with different as the contained sequence of sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable, but have kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the aminoacid sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding is with identical at least about 60% as the sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable, more preferably with identical at least about 70% as one of sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable, even more preferably with as the sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable at least about 80%, 90% or 95% homology, and most preferably with identical at least about 96%, 97%, 98% or 99% as the sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table II B the 7th row, 72-81 and 460-462 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 72-81 with 460-462 is capable, preferred Table II B the 7th row, 72-81 and the 460-462 aminoacid sequence shown in capable is at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 72-81 and 460-462 are capable, preferred Table II B the 7th row, 72-81 is identical at least about 60% with the sequence shown in 460-462 is capable, more preferably with as Table II the 5th or 7 be listed as, 72-81 and 460-462 are capable, preferred Table II B the 7th row, 72-81 is identical at least about 70% with one of sequence shown in 460-462 is capable, even more preferably with as Table II the 5th or 7 be listed as, 72-81 and 460-462 are capable, preferred Table II B the 7th row, sequence shown in 72-81 and 460-462 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 72-81 and 460-462 are capable, preferred Table II B the 7th row, sequence shown in 72-81 and 460-462 are capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.0] was to [0175.0.0.0] above [0169.0.0.7] to [0175.0.5.7] saw for disclosing of [0169.0.0.7] to [0175.0.5.7] these paragraphs

[0176.0.7.7] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in 72-81 and 460-462 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 72-81 and 460-462 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in 72-81 and 460-462 are capable has essentially identical character and discerns.

[0177.0.7.7] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the functional equivalent that nucleotide sequence obtained shown in 72-81 and 460-462 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 72-81 and 460-462 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in 72-81 and 460-462 are capable.

[0178.0.0.7] sees [0178.0.0.0] for disclosing of this paragraph

[0179.0.7.7] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I the 5th or 7 row, 72-81 and 460-462 capable shown in), and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table II B the 7th is listed as, 72-81 and 460-462 capable shown in the nucleic acid molecule of homologous protein of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to encoding sequence, in sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable, introduce sudden change.

[0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.7] to [0183.0.0.7] saw for disclosing of [0180.0.0.7] to [0183.0.0.7] these paragraphs

[0184.0.7.7] is employed to have as Table I the 5th or 7 row, 72-81 and 460-462 are capable, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in 72-81 and 460-462 are capable, perhaps come Table II the 5th or 7 row freely, 72-81 and 460-462 are capable, preferred Table II B the 7th row, the homologous compound of the nucleotide sequence of sequence shown in 72-81 and 460-462 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.7.7] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I the 5th or 7 row, 72-81 with 460-462 is capable, preferred Table I B the 7th row, 72-81 and the 460-462 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 72-81 with 460-462 is capable, other nucleotide sequence of not showing in preferred Table I B the 7th row, 72-81 and the 460-462 arbitrary sequence shown in capable.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 72-81 with 460-462 is capable, preferred Table I B the 7th row, 72-81 and the 460-462 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.7.7] also preferred the inventive method comprise as Table II the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table II B the 7th row, 72-81 and 460-462 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table II B the 7th row, 72-81 and 460-462 capable shown in sequence identical.

[0187.0.7.7] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table II B the 7th row, 72-81 and 460-462 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table II B the 7th row, 72-81 and 460-462 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.7.7] still has the basic biologic activity of polypeptide of the present invention (being its active basic reduction that do not have) of giving fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active comparing with polypeptide expressed activity as Table II the 5th or 7 row, 72-81 and shown in 460-462 is capable and under the same conditions do not reduced substantially.In one embodiment, polypeptide of the present invention is to comprise as sequence shown in Table II B the 7th row, 72-81 and 460-462 are capable or by its homologue of forming.

[0189.0.7.7] as the homologous compound of sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.7], [0191.0.5.7], [00191.1.0.7] and [0192.0.0.7] to [0203.0.0.7] is for [0190.0.0.7], [0191.0.5.7], [0191.1.0.7] and [0192.0.0.7] is above disclosing of [0203.0.0.7] these paragraphs seen [0190.0.0.0], [0191.0.0.0], [0191.1.0.0] and [0192.0.0.0] is to [0203.0.0.0]

[0204.0.7.7] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table II B the 7th row, 72-81 and 460-462 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given fine chemicals quantity in biological or its part increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 7th row, 72-81 and 460-462 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as the primer shown in Table III the 7th row, 72-81 and 460-462 are capable or primer amplification from cDNA library or genomic library is obtained by using, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 72-81 and 460-462 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described peptide coding is as Table II the 5th or 7 row, 72-81 and 460-462 is capable, the structural domain of preferred Table II B the 7th row, 72-81 and the 460-462 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, nucleic acid molecule shown in 72-81 and 460-462 are capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 72-81 and 460-462 are capable

The fragment of preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt; Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A the 5th or 7 row, 72-81 and 460-462 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule do not form by Table I A or IB the 5th or 7 row, 72-81 and 460-462 by the sequence shown in capable.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 72-81 and 460-462 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as the peptide sequence shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as the protein of sequence shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable at least 30% identical and with Table II A or IIB the 5th or 7 row, 72-81 and 460-462 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.0] was to [0226.0.0.0] above [0205.0.0.7] to [0226.0.0.7] saw for disclosing of [0205.0.0.7] to [0226.0.0.7] these paragraphs

[0227.0.7.7] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 72-81 and the capable sequence or derivatives thereof of mentioning of 460-462, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid biosynthetic pathway of at least one lipid acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases that synthetic each is amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 72-81 and 460-462 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.0] was to [0230.0.0.0] above [0228.0.5.7] to [0230.0.5.7] saw for disclosing of [0228.0.5.7] to [0230.0.5.7] these paragraphs

[0231.0.7.7] in another advantageous embodiment of the inventive method, the employed biology of present method is to have weakened the stearic protein of degraded simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.7] to [0276.0.0.7] saw for disclosing of [0232.0.0.7] to [0276.0.0.7] these paragraphs

[0277.0.5.7] sees [0277.0.5.5] for disclosing of this paragraph

[0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.7] to [0282.0.0.7] saw for disclosing of [0278.0.0.7] to [0282.0.0.7] these paragraphs

[0283.0.7.7] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-antibody as polypeptide shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or its antigen part, polypeptide (polypeptide for example of the present invention or its fragment) generation that it can comprise above-mentioned sequence or be made up of above-mentioned sequence by the standard technique utilization.Preferably specificity is in conjunction with the monoclonal antibody as polypeptide shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable.

[0284.0.0.7] sees [0284.0.0.0] for disclosing of this paragraph

[0285.0.7.7] the present invention relates to have as sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable in one embodiment.

[0286.0.7.7] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in 72-81 and 460-462 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in 72-81 and 460-462 are capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to and comprising more than a polypeptide as consensus sequence shown in Table IV the 7th row, 72-81 and 460-462 are capable.

[0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.7] to [0290.0.0.7] saw for disclosing of [0287.0.0.7] to [0290.0.0.7] these paragraphs

[0291.0.7.7] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise and contain plant or microorganism specificity consensus sequence or by its polypeptide of forming.In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in 72-81 and 460-462 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in 72-81 and 460-462 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable.

[0292.0.0.7] sees [0292.0.0.0] for disclosing of this paragraph

[0293.0.7.7] the present invention relates to give the polypeptide that each fine chemicals increases in one embodiment in biological or its part, described polypeptide is by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention coding.

In one embodiment, polypeptide of the present invention have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help to form as the sequence of nucleic acid molecule encoding shown in Table I A or IB the 5th or 7 row, 72-81 and 460-462 are capable.

[0294.0.7.7] in one embodiment, the present invention relates to have as Table II the 3rd row, the polypeptide of activity of proteins shown in 72-81 and 460-462 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in 72-81 and 460-462 are capable.

[0295.0.0.7], [0296.0.0.7] and [0297.0.5.7] are for [0295.0.0.7], and disclosing of [0296.0.0.7] and [0297.0.5.7] these paragraphs sees that top [0295.0.0.0] is to [0297.0.0.0]

The chemical of [00297.1.0.7] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 72-81 and 460-462 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.

[0298.0.7.7] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II the 5th or 7 row, 72-81 and 460-462, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable.

[0299.0.7.7] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of aminoacid sequence shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable.

[0300.0.7.7] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,72-81 and 460-462 capable shown in sequence different.Therefore, this polypeptide contains with complete amino acid sequence homology as sequence shown in Table II A or IIB the 5th or 7 row, 72-81 and 460-462 are capable and is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.7] sees [0301.0.0.0] for disclosing of this paragraph

The biologically-active moiety of [0302.0.7.7] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence of the aminoacid sequence shown in 72-81 and 460-462 are capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.7] sees [0303.0.0.0] for disclosing of this paragraph

[0304.0.7.7] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as the active of polypeptide shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.5.7], [0306.0.5.7] and [0306.1.0.7] are for [0305.0.5.7], and top [0305.0.0.0], [0306.0.0.0] and [0306.1.0.0] are seen in disclosing of [0306.0.5.7] and [0306.1.0.7] these paragraphs

Above [0307.0.0.7] and [0308.0.0.7] sees for disclosing of [0307.0.0.7] and [0308.0.0.7] these paragraphs [0307.0.0.0and[0308.0.0.0]

[0309.0.7.7] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned, as Table II the 5th or 7 row, be meant polypeptide shown in 72-81 and 460-462 are capable with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " non-polypeptide " during 72-81 and 460-462 are capable or " other polypeptide " are meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with the homology not basically of the polypeptide with protein active, for example do not give described in the literary composition active and from the protein of identical or different biology.

[0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.7] to [0334.0.0.7] saw for disclosing of [0310.0.0.7] to [0334.0.0.7] these paragraphs

[0335.0.7.7] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of one of sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.7] to [0342.0.0.7] saw for disclosing of [0336.0.0.7] to [0342.0.0.7] these paragraphs

[0343.0.7.7] as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 72-81 and 460-462 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as one of sequence shown in Table I the 5th or 7 row, 72-81 and 460-462 are capable or its homologue in a kind of organism.

[0344.0.0.7] is to [0350.0.0.7], [0351.0.5.7] and [0352.0.0.7] to [0361.0.0.7] for [0344.0.0.7] to [0350.0.0.7], [0351.0.5.7] and [0352.0.0.7] above disclosing of [0361.0.0.7] these paragraphs seen [0344.0.0.0] to [0361.0.0.0]

[0362.0.7.7] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or each fine chemicals of its part increases) carry out genetically modified any cell, the nucleic acid molecule of described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide (for example coding has the polypeptide of protein as polypeptide active shown in Table II the 3rd row, 72-81 and 460-462 are capable).Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, cytoactive improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Transgenosis with the active polypeptide of protein (as polypeptide shown in Table II the 3rd row, 72-81 and 460-462 are capable) is meant in the text because genomic regulation and control or manipulation, improves having protein (as Table II the 3rd row, 72-81 and polypeptide shown in 460-462 is capable) activity active or similar active polypeptide in cell or biological or its part.Example and the inventive method are described in above.

[0363.0.0.7], [0364.0.5.7] and [0365.0.0.7] to [0379.0.5.7 is for [0363.0.0.7], [0364.0.5.7] and [0365.0.0.7] above disclosing of [0379.0.5.7] these paragraphs seen [0363.0.0.0] to [0379.0.0.0]

[0380.0.5.7], [0381.0.0.7] and [0382.0.0.7] are for [0380.0.5.7], and top [0380.0.5.5], [0381.0.0.0] and [0382.0.0.0] are seen in disclosing of [0381.0.0.7] and [0382.0.0.7] these paragraphs

[0383.0.5.7], [0384.0.0.7], [0385.0.5.7] and [0386.0.5.7] is for [0383.0.5.7], [0384.0.0.7], top [0383.0.5.5] seen in disclosing of [0385.0.5.7] and [0386.0.5.7] these paragraphs, [0384.0.0.0], [0385.0.5.5] and [0386.0.5.5]

[0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.7] to [0392.0.0.7] saw for disclosing of [0387.0.0.7] to [0392.0.0.7] these paragraphs

[0393.0.7.7] the present invention relates to identify and gives the method that the compound that becomes more meticulous in the cell produces the gene product that increases that it comprises following step in one embodiment:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 72-81 and 460-462 is capable, preferred Table I B the 5th or 7 row, 72-81 and the 460-462 nucleic acid molecule shown in capable is hybridized under lax stringent condition nucleic acid molecule, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.0] was to [0416.0.0.0] above [0394.0.0.7] to [0415.0.0.7] and [0416.0.5.7] saw for disclosing of [0394.0.0.7] to [0415.0.0.7] and [0416.0.5.7] these paragraphs

[0417.0.5.7] and [0418.0.0.7] to [0430.0.0.7] for [0417.0.5.7] and [0418.0.0.7] above disclosing of [0430.0.0.7] these paragraphs seen [0417.0.5.5] and [0418.0.0.0] to [0430.0.0.0]

[0431.0.5.7], [0432.0.5.7], [0433.0.0.7] and [0434.0.0.7] be for [0431.0.5.7], and [0432.0.5.7], [0431.0.0.0] was to [0434.0.0.0] above the disclosing of [0433.0.0.7] and [0434.0.0.7] these paragraphs seen

[0435.0.5.5] was to [0440.0.5.5] above [0435.0.5.7] to [0440.0.5.7] saw for disclosing of [0435.0.5.7] to [0440.0.5.7] these paragraphs

Above [0441.0.0.7] and [0442.0.5.7] sees for disclosing of [0441.0.0.7] and [0442.0.5.7] these paragraphs [0441.0.0.0] and [0442.0.5.5]

[0443.0.0.7] sees [0443.0.0.0] for disclosing of this paragraph

Above [0444.0.5.7] and [0445.0.5.7] sees for disclosing of [0444.0.5.7] and [0445.0.5.7] these paragraphs [0444.0.5.5] and [0445.0.5.5]

[0446.0.0.0] was to [0453.0.0.0] above [0446.0.0.7] to [0453.0.0.7] saw for disclosing of [0446.0.0.7] to [0453.0.0.7] these paragraphs

Above [0454.0.5.7] and [0455.0.5.7] sees for disclosing of [0454.0.5.7] and [0455.0.5.7] these paragraphs [0454.0.5.5] and [0455.0.5.5]

[0456.0.0.7] sees [0456.0.0.0] for disclosing of this paragraph

[0457.0.5.5] was to [0460.0.5.5] above [0457.0.5.7] to [0460.0.5.7] saw for disclosing of [0457.0.5.7] to [0460.0.6.7] these paragraphs

[0461.0.7.7] embodiment 10: clone SEQ ID NO:5818 is used for expressing plant

[0462.0.0.7] sees [0462.0.0.0] for disclosing of this paragraph

[0463.0.7.7] passes through pcr amplification SEQ ID NO:5818 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.5.7], [0465.0.5.7] and [0466.0.0.7] are for [0464.0.5.7], and top [0464.0.5.5], [0465.0.5.5] and [0466.0.0.0] are seen in disclosing of [0465.0.5.7] and [0466.0.0.7] these paragraphs

[0467.0.7.7] selects following primer sequence for genes of SEQ ID NO:5818:

I) forward primer (SEQ ID NO:6426)

ttatttttcc?tgaagaccga?gttttt

Ii) reverse primer (SEQ ID NO:6427)

atgctggata?aaattgttat?tgcc

[0468.0.0.0] was to [0479.0.0.0] above [0468.0.0.7] to [0479.0.0.7] saw for disclosing of [0468.0.0.7] to [0479.0.0.7] these paragraphs

[0480.0.7.7] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:5818

[0481.0.0.7] sees [0481.0.0.0] for disclosing of this paragraph

[0482.0.0.0] was to [0513.0.0.0] above [0482.0.0.7] to [0513.0.0.7] saw for disclosing of [0482.0.0.7] to [0513.0.0.7] these paragraphs

[0514.0.7.7] selects as another kind of, as people such as Geigenberger (Plant Cell﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect amino acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??b3256	Stearic acid (C18:0)	??GC	??1.17	??1.25
??b2699	Stearic acid (C18:0)	??GC	??1.22	??1.83
					??b2095	Stearic acid (C18:0)	??GC	??1.17	??1.26
??YOR024W	Stearic acid (C18:0)	??GC	??1.16	??1.40
					??YBR089C-A	Stearic acid (C18:0)	??GC	??1.36	??2.34
??YFR042W	Stearic acid (C18:0)	??GC	??1.19	??1.26
					??YIL150C	Stearic acid (C18:0)	??GC	??1.34	??3.20
??YDR513W	Stearic acid (C18:0)	??GC	??1.16	??1.51
					??YLR010C	Stearic acid (C18:0)	??GC	??1.16	??1.76

??ORF	Metabolite	Method	??Min	??Max
					??b1093	Stearic acid (C18:0)	??GC	??1.16	??1.31
??b0161	Stearic acid (C18:0)	??GC	??1.16	??2.29
					??b1896	Stearic acid (C18:0)	??GC	??1.23	??1.30
??b3457	Stearic acid (C18:0)	??GC	??1.18	??1.34

[0515.0.5.7] the 2nd row have shown the lipid acid of being analyzed.The 4th row and the 5th row have shown the ratio of lipid acid between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

Above [0516.0.0.7] and [0517.0.5.7] sees for disclosing of [0516.0.0.7] and [0517.0.5.7] these paragraphs [0516.0.0.0] and [0517.0.0.0]

[0518.0.0.0] was to [0530.0.0.0] above [0518.0.0.7] to [0529.0.0.7] and [0530.0.5.7] saw for disclosing of [0518.0.0.7] to [0529.0.0.7] and [0530.0.5.7] these paragraphs

[0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.5] to [0530.6.0.5] saw for disclosing of [0530.1.0.5] to [0530.6.0.5] these paragraphs

[0531.0.0.0] was to [0534.0.0.0] above [0531.0.0.7] to [0533.0.0.7] and [0534.0.5.7] saw for disclosing of [0531.0.0.7] to [0533.0.0.7] and [0534.0.5.7] these paragraphs

[0535.0.0.0] was to [0538.0.0.0] above [0535.0.0.7] to [0537.0.0.7] and [0538.0.5.7] saw for disclosing of [0535.0.0.7] to [0537.0.0.7] and [0538.0.5.7] these paragraphs

[0539.0.0.0] was to [0543.0.0.0] above [0539.0.0.7] to [0542.0.0.7] and [0543.0.5.7] saw for disclosing of [0539.0.0.7] to [0542.0.0.7] and [0543.0.5.7] these paragraphs

[0544.0.0.0] was to [0552.0.0.0] above [0544.0.0.7] to [0547.0.0.7] and [0548.0.5.7] saw for disclosing of [0544.0.0.7] to [0547.0.0.7] and [0548.0.5.7] to [0552.0.0.7] these paragraphs to [0552.0.0.7]

[0552.1.0.7]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF title	Metabolite	??Min	??Max
				??YIL150C	Stearic acid (C18:0)	??1.469	??2.241

Table 2 shows that stearic acid has increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YIL150c.

In one embodiment, yeast saccharomyces cerevisiae protein YIL150C or its homologue in maize plant, for example under the situation that the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue is enhanced, preferably, giving the fine chemicals stearic acid is increased between 46% and 124%.

[0552.2.0.7] sees [0552.2.0.0] for disclosing of this paragraph

[0553.0.7.7]

1. produce stearic method, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable or its function equivalent; With

(b) in allowing described biology, produce this biology of cultivation under the stearic condition.

2. produce stearic method, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable, and described nucleic acid molecule gives that stearic acid quantity increases in biology or its part;

B) contain just like Table I the 5th or 7 row, 72-81 and 460-462 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of stearic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of stearic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that stearic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 72-81 and 460-462 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that stearic acid quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that stearic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 72-81 and 460-462 capable shown in consensus sequence polypeptide and give biology or its part in stearic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give stearic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded stearic acid.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded stearic acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 72-81 and 460-462 are capable, and described nucleic acid molecule gives that stearic acid quantity increases in biology or its part;

B) contain just like Table I the 5th or 7 row, 72-81 and 460-462 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of stearic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of stearic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that stearic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 72-81 and 460-462 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that stearic acid quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that stearic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 72-81 and 460-462 capable shown in consensus sequence polypeptide and give biology or its part in stearic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give stearic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A the 5th or 7 row, 72-81 and 460-462 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 72-81 and 460-462 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in stearic acid quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part stearic acid quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps stearic acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the stearic acid level that will measure or expression of polypeptides level and standard stearic acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the stearic acid generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of stearic acid quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of stearic acid quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the stearic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that stearic acid increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the suitable stearic host cell that produces;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) the stearic acid level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give stearic acid level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the stearic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that stearic acid quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the suitable stearic host cell that produces;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) the stearic acid level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give stearic acid level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that stearic acid increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control stearic acid level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make the anti-stearic acid synthetic weedicide that suppresses of plant.

[0554.0.0.7] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.8] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.8] and [0002.0.7.8] sees the previous paragraph [0001.0.0.0] and [0002.0.7.7] for disclosing of paragraph [0001.0.0.8] and [0002.0.7.8].

[0003.0.8.8] palmitinic acid is a main component of making soap, lubricating oil and water-proof material.In addition, it can be used for synthetic palmitinic acid metal-salt.Other purposes is as foodstuff additive and is used for synthesised food level additive, and the moiety that is used as cosmetic formulations.Palmitinic acid is the natural fat and the oils of many glyceride forms, for example palmitic main component, and be the main component of most of commercial grade stearic acid products.

Above [0004.0.7.8] and [0005.0.5.8] sees for disclosing of [0004.0.7.8] and [0005.0.5.8] these paragraphs [0004.0.7.7] and [0005.0.5.5]

[0006.0.8.8] as mentioned above, palmitinic acid is the main fat in meat and the milk preparation.

The further purposes of [0007.0.8.8] palmitinic acid is as the agent of food composition emulsifying raw material, perhaps as face cream and the personal care emulsifying agent of washing one's face and revealing.Palmitinic acid is also as shaving cream preparation, wax or fruit wax preparation.

[0008.0.8.8] palmitinic acid is also as shaving cream preparation, wax or fruit wax preparation.

Above [0009.0.7.8] to [0012.0.7.8] sees for disclosing of [0009.0.7.8] to [0012.0.7.8] these paragraphs [0009.0.7.7] and [0012.0.7.7]

[0013.0.0.8] sees [0013.0.0.0] for disclosing of this paragraph

[0014.0.8.8] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is palmitinic acid or the triglyceride level that contains palmitinic acid, lipid, oils or fat.Therefore, in the present invention, term " fine chemicals " is meant " palmitinic acid and/or contain triglyceride level, lipid, oils or the fat of palmitinic acid " as used herein.In addition, term " fine chemicals " also refers to comprise palmitinic acid and/or contains the triglyceride level, lipid, oils of palmitinic acid or the fine chemicals of fat as used herein.

[0015.0.8.8] an embodiment, term " fine chemicals " meaning is meant palmitinic acid and/or contains triglyceride level, lipid, oils or the fat of palmitinic acid.In whole specification sheets, term " fine chemicals " meaning is meant palmitinic acid and/or contains triglyceride level, lipid, oils or fat, palmitinic acid or its salt, ester, thioesters or the free form of palmitinic acid or in conjunction with the palmitinic acid of other compound such as triacylglycerol, glycolipid, phosphatide etc.In preferred embodiments, term " fine chemicals " meaning is meant free palmitinic acid or its salt or is bonded to the palmitinic acid of triacylglycerol.Triacylglycerol, lipid, oils, fat or its lipid mixtures should refer to any triacylglycerol, lipid, oils and/or contain the fat of any combination or free palmitinic acid, for example sphingolipid, phosphoglyceride, lipid, glycolipid class such as sphingoglycolipid, phosphatide such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, glycerine Serine, phosphatidylinositols or two glycerine Serines or monoacylglycerol, diacylglycerol or triacylglycerol or other fatty acid ester such as acetyl-CoA thioesters, it contains other saturated or unsaturated fatty acids in fatty acid molecule.

In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning be meant to have the active at least a chemical compound of above-mentioned fine chemicals.

[0016.0.8.8] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce protein YDR513W, YDR447C, YBR089C-A, b3256, YGR126W, YPL099C, b0399, b0849, b3457, b3578, b3644 and/or b4129 or have by Table I the 5th or 7 row, 82-88 and 463-467 capable shown in the activity of proteins of the coded peptide sequence of nucleic acid molecule; With

(b) in allowing described biology, produce under the condition that fine chemicals is palmitinic acid or the fine chemicals that comprises palmitinic acid and make biological growth.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II the 3rd row, 82-88 and 463-467 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, 82-88 and 463-467 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) under the condition that allows generation fine chemicals, particularly palmitinic acid, make biological growth.

Above [0017.0.0.8] and [0018.0.0.8] sees for disclosing of [0017.0.0.8] and [0018.0.0.8] these paragraphs [0017.0.0.0] and [0018.0.0.0]

The method that [0019.0.8.8] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare the fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 82-88 and 463-467 are capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 82-88 and 463-467 were capable.

[0020.0.8.8] be surprisingly found out that, at least a shown in Table II the 3rd row, 82-84 and 86 and 87 row yeast saccharomyces cerevisiae protein and/or at least a such as Table II the 3rd row, the 85th and 88 and e. coli k12 protein in Arabidopis thaliana the transgene expression of 463-467 shown in capable give institute's conversion plant palmitinic acid (or fine chemicals) content raising.

[0021.0.0.8] sees [0021.0.0.0] for disclosing of this paragraph

The sequence of [0022.0.8.8] e. coli k12 b3256 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be acetyl CoA carboxylase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary acetyl-CoA carboxylase or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of acetyl-CoA carboxylase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary acetyl-CoA carboxylase or its homologue is enhanced or produces.

The sequence of e. coli k12 b0399 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the positive reaction instrumentality of pho regulon.Therefore, in one embodiment, the inventive method comprises as shown here from the positive reaction instrumentality of colibacillary pho regulon or the purposes of its homologue, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of the positive reaction instrumentality of pho regulon is enhanced or produces in the methods of the invention, for example is enhanced or produces from the positive reaction instrumentality of colibacillary pho regulon or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YDR513W has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be described as be glutaredoxin (thiol transferase, glutathione reductase).Therefore, in one embodiment, the inventive method comprises the purposes of glutaredoxin or its homologue from yeast saccharomyces cerevisiae as shown here, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of glutaredoxin is enhanced or produces in the methods of the invention, for example is enhanced or produces from the glutaredoxin of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YDR447C has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive be described as be " small subunit ribosome (40s) protein 51 (rp51); Almost identical and have similarity with rat rat S17 ribosomal protein with rps17Ap; Rps17bp ".Therefore, in one embodiment, the inventive method comprises the purposes of the small subunit ribosome from yeast saccharomyces cerevisiae as shown here (40s) protein 51 (rp51) or its homologue, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of small subunit ribosome (40s) protein 51 (rp51) is enhanced or produces in the methods of the invention, for example is enhanced or produces from small subunit ribosome (40s) protein 51 (rp51) of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YBR089C-A has been published in Feldmann etc., EMBO J., and 13 (24), 5795-5809 (1994) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.It shows with Mammals high mobility group protein 1 and 2 to have homology.Its function may be unnecessary to height homologous gene NHP6A.In addition, it shows and the nonhistones chromatin protein N hp6bp homology of high speed swimming family.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YBR089C-A from yeast saccharomyces cerevisiae as shown here, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the YBR089C-A activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YBR089C-A protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YGR126W has been published in Tettelin etc., Nature 387 (6632 supplementary issue), and 81-84 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.It is " open reading-frame (ORF) of supposition " seemingly.Therefore, in one embodiment, the inventive method comprises the purposes of active or its homologue of YGR126W from yeast saccharomyces cerevisiae as shown here, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the YGR126W activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YGR126W protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YPL099C has been published in Bussey etc., Nature 387 (6632 supplementary issue), and 103-105 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized." believable untagged protein positioning is in plastosome ".Therefore, in one embodiment, the inventive method comprises the purposes of YPL099C or its homologue from yeast saccharomyces cerevisiae as shown here, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the YPL099C activity of proteins is enhanced or produces in the methods of the invention, for example is enhanced or produces from the YPL099C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0849 (accession number NP_415370) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the glutaredoxin 1 redox coenzyme of gsh dependency ribonucleotide reductase.Therefore, in one embodiment, the inventive method comprises as shown here from the glutaredoxin 1 redox cofactor protein of colibacillary gsh dependency ribonucleotide reductase or the purposes of its homologue, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of the glutaredoxin 1 redox cofactor protein of gsh dependency ribonucleotide reductase is enhanced or produces in the methods of the invention, for example is enhanced or produces from the glutaredoxin 1 redox cofactor protein of colibacillary gsh dependency ribonucleotide reductase or the activity of its homologue.

The sequence of e. coli k12 b3457 (accession number NP_417914) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary high-affinity branched-chain amino acid translocator or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of high-affinity branched-chain amino acid translocator is enhanced or produces in the methods of the invention, and for example the activity from colibacillary high-affinity branched-chain amino acid translocator or its homologue is enhanced or produces.

The sequence of e. coli k12 b3578 (accession number YP_026232) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the movement system composition.Therefore, in one embodiment, the inventive method comprises as shown here from the movement system composition of colibacillary supposition or the purposes of its homologue, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of Jia Ding movement system composition is enhanced or produces in the methods of the invention, for example is enhanced or produces from the movement system composition of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b3644 (accession number NP_418101) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the stress-induced protein that does not characterize.Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary stress-induced protein that does not characterize or the purposes of its homologue, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the stress-induced activity of proteins that does not characterize is enhanced or produces in the methods of the invention, for example is enhanced or produces from the colibacillary stress-induced protein that does not characterize or the activity of its homologue.

The sequence of e. coli k12 b4129 (accession number NP_418553) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Methionin tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary Methionin tRNA synthetase protein or its homologue as shown here, as described its be used for biological or its part produces fine chemicals, be palmitinic acid and/or the triglyceride level that contains palmitinic acid, lipid, oils and/or fat, particularly increase palmitinic acid and/or contain triglyceride level, lipid, oils and/or the fat of palmitinic acid, the preferred quantity of the palmitinic acid of free or combining form.In one embodiment, the activity of Methionin tRNA synthetase protein is enhanced or produces in the methods of the invention, and for example the activity from colibacillary Methionin tRNA synthetase protein or its homologue is enhanced or produces.

The homologous compound (=homologue) of [0023.0.8.8] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.

Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with protein shown at least a Table II the 3rd row, 82-88 and 463-467 are capable, for example have by contain Table I the 5th or 7 row, 82-88 and 463-467 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in Table II 82-84 and 86 and 87 row in the polypeptide homologue of any one be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in the biology, and described homologue is from eukaryote.In one embodiment, Table II the 3rd row, the 85th and 88 and 463-467 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from bacterium.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 82-84 and 86 and 87 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from fungi.In one embodiment, Table II the 3rd row, the 85th and 88 and 463-467 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Proteobacteria.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 82-84 and 86 and 87 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Ascomycota.In one embodiment, Table II the 3rd row, the 85th and 88 and 463-467 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 82-84 and 86 and 87 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from yeast.In one embodiment, Table II the 3rd row, the 85th and 88 and 463-467 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the enterobacteria order.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 82-84 and 86 and 87 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.In one embodiment, Table II the 3rd row, the 85th and 88 and 463-467 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from enterobacteriaceae.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 82-84 and 86 and 87 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetes.In one embodiment, Table II the 3rd row, the 85th and 88 and 463-467 capable shown in the homologue of polypeptide be to have same or similar active homologue, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Escherichia.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 82-84 and 86 and 87 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from Saccharomycetaceae.In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 82-84 and 86 and 87 row, particularly active increasing given fine chemicals content increase in biology or its part, and described homologue is from the yeast guiding principle.

Above [0023.1.0.8] and [0024.0.0.8] sees for disclosing of [0023.1.0.8] and [0024.0.0.8] these paragraphs [0023.1.0.0] and [0024.0.0.0]

[0025.0.8.8] is according to the present invention, if triglyceride level, lipid, oils or fat level that from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause palmitinic acid in biology or its part, the preferred described biomass cells and/or contain palmitinic acid increase and protein has as proteinic above-mentioned activity shown in Table II the 3rd row, 82-88 and 463-467 are capable, then described protein or polypeptide have " as activity of proteins shown in Table II the 3rd row, 82-88 and 463-467 are capable ".In this manual, if this kind protein or polypeptide still have Table II the 3rd row, proteinic biology or the enzyme activity shown in 82-88 and 463-467 are capable, if promptly with Table II the 3rd row, 82-84 compares with yeast saccharomyces cerevisiae protein shown in 87 row with 86 and/or is listed as with Table II the 3rd, the 85th compares with e. coli k12 protein shown in 463-467 is capable with 88, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

Above [0025.1.0.8] and [0025.2.0.8] sees for disclosing of [0025.1.0.8] and [0025.2.0.8] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.8] to [0033.0.0.8] saw for disclosing of [0026.0.0.8] to [0033.0.0.8] these paragraphs

[0034.0.8.8] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 82-88 and protein shown in 463-467 is capable or by the active protein expression level of the protein of Table I the 5th row, 82-88 and nucleic acid molecule encoding shown in 463-467 is capable or its homologue (as Table I the 7th row, 82-88 and homologue shown in 463-467 is capable) or active aspect different, and it is difference aspect biological chemistry or genetics reason, and therefore shows the fine chemicals quantity that increases.

[0035.0.0.0] was to [0039.0.0.0] above [0035.0.0.8] to [0038.0.0.8] and [0039.0.5.8] saw for disclosing of [0035.0.0.8] to [0038.0.0.8] and [0039.0.5.8] these paragraphs

Above [0040.0.0.8] to [0044.0.0.8] sees for disclosing of [0040.0.0.8] to [0044.0.0.8] these paragraphs [0035.0.0.0] and [0044.0.0.0]

[0045.0.8.8] in one embodiment, at e. coli k12 protein b3256 or its homologue, for example under the activity situation about being enhanced of acetyl-CoA carboxylase protein (for example shown in Table II the 5th or 7 row, the 85th row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 15% and 17% or more between.

At e. coli k12 protein b0399 or its homologue, for example under the activity situation about being enhanced of the positive reaction instrumentality of pho regulon (for example shown in Table II the 5th or 7 row, the 88th row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 20% and 27% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR513W or its homologue, under the activity situation about being enhanced of for example " glutaredoxin " (for example shown in Table II the 5th or 7 row, the 82nd row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 17% and 74% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR447C or its homologue, under the activity situation about being enhanced of for example " small subunit ribosome (40s) protein 51 (rp51) " (for example shown in Table II the 5th or 7 row, the 83rd row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 19% and 181% or more between.

At yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, under the activity situation about being enhanced of for example " not profiling protein matter YBR089C-A " (it " has homology with Mammals high mobility group protein 1 and 2 ") (for example shown in Table II the 5th or 7 row, the 84th row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 55% and 95% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YGR126W or its homologue, under the activity situation about being enhanced of for example " not profiling protein matter YGR126W " (for example shown in Table II the 5th or 7 row, the 86th row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 34% and 167% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPL099C or its homologue, under the activity situation about being enhanced of for example " not profiling protein matter YPL099C " (for example shown in Table II the 5th or 7 row, the 87th row), preferably, in one embodiment, give fine chemicals, preferred of palmitinic acid be increased in 21% and 55% or more between.

In one embodiment, at e. coli k12 protein b0849 or its homologue, for example under the activity situation about being enhanced of the glutaredoxin 1 redox cofactor protein of gsh dependency ribonucleotide reductase (for example shown in Table II the 5th or 7 row, the 463rd row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 17% and 30% or more between.

In one embodiment, at e. coli k12 protein b3457 or its homologue, for example under the activity situation about being enhanced of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) (for example shown in Table II the 5th or 7 row, the 464th row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 16% and 36% or more between.

In one embodiment, at e. coli k12 protein b3578 or its homologue, for example under the activity situation about being enhanced of Jia Ding movement system composition (for example shown in Table II the 5th or 7 row, the 465th row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 16% and 27% or more between.

In one embodiment, at e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of the stress-induced protein that does not characterize (for example shown in Table II the 5th or 7 row, the 466th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 16% and 42% or more between.

In one embodiment, at e. coli k12 protein b4129 or its homologue, for example under the activity situation about being enhanced of Methionin tRNA synthetic enzyme (for example shown in Table II the 5th or 7 row, the 467th row), preferably, give in one embodiment fine chemicals, preferred palmitinic acid be increased in 33% and 36% or more between.

[0046.0.8.8] is at e. coli k12 protein b3256 or its homologue, for example under the situation that the acetyl-CoA carboxylase activity of proteins is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

At e. coli k12 protein b0399 or its homologue, for example under the situation that the positive reaction instrumentality activity of proteins of pho regulon is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

At yeast saccharomyces cerevisiae protein YDR513W or its homologue, for example under the situation that the activity of " glutaredoxin " is enhanced, preferably, in one embodiment, triglyceride level, lipid, oils and/or the fat of give fine chemicals, preferably giving fine chemicals and contain palmitinic acid increase.

At yeast saccharomyces cerevisiae protein YDR447C or its homologue, for example under the situation that the activity of " the necessary small subunit ribosome of cell survival (40s) protein 51 (rp51) " is enhanced, preferably, in one embodiment, triglyceride level, lipid, oils and/or the fat of give fine chemicals, preferably giving fine chemicals and contain palmitinic acid increase.

At yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, under the situation that the activity of for example " not profiling protein matter YBR089C-A " (it " has homology with Mammals high mobility group protein 1 and 2 ") is enhanced, preferably, giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

At yeast saccharomyces cerevisiae protein YGR126W or its homologue, for example under the situation that the activity of " not profiling protein matter YGR126W " is enhanced, preferably, in one embodiment, triglyceride level, lipid, oils and/or the fat of give fine chemicals, preferably giving fine chemicals and contain palmitinic acid increase.

At yeast saccharomyces cerevisiae protein YPL099C or its homologue, for example under the situation that the activity of " not profiling protein matter YPL099C protein " is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b0849 or its homologue, for example under the situation that the activity of the glutaredoxin 1 redox cofactor protein of gsh dependency ribonucleotide reductase is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b3457 or its homologue, for example under the situation that the activity of high-affinity branched-chain amino acid translocator (ATP-binding cassette superfamily) is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b3578 or its homologue, for example under the situation that the activity of Jia Ding movement system composition is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b3644 or its homologue, for example under the situation that the stress-induced activity of proteins of Biao Zhenging is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

In one embodiment, at e. coli k12 protein b4129 or its homologue, for example under the situation that the activity of Methionin tRNA synthetic enzyme is enhanced, preferably giving fine chemicals increases with the triglyceride level that contains palmitinic acid, lipid, oils and/or fat.

Above [0047.0.0.8] to [0048.0.0.8] sees for disclosing of [0047.0.0.8] and [0048.0.0.8] these paragraphs [0047.0.0.0] and [0048.0.0.0]

[0049.0.8.8] has to give and improves each fine chemicals quantity or the active protein of level preferably has polypeptide described herein, particularly be listed as comprising described in the literary composition as Table IV the 7th, the polypeptide of consensus sequence shown in 82-88 and 463-467 are capable, perhaps as Table II the 5th and 7 row, its function homologue described in polypeptide or the literary composition shown in the capable disclosed aminoacid sequence of 82-88 and 463-467, perhaps (for example be listed as by Table I the 5th and 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 82-88 and 463-467 are capable or its function homologue as herein described) structure of coded polypeptide, and have the activity described in the literary composition.

[0050.0.8.8] for the purposes of the present invention, term " palmitinic acid " also comprises corresponding salt, for example, the sylvite of palmitinic acid or sodium salt or have amine such as the salt of the palmitinic acid of diethylamine.

Above [0051.0.5.8] and [0052.0.0.8] sees for disclosing of [0051.0.5.8] and [0052.0.0.8] these paragraphs [0051.0.0.0] and [0052.0.0.0]

[0053.0.8.8] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having as the active polypeptide of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 7th row, 82-88 and 463-467 capable shown in)) and is expressed and increase, and has the activity of the raising fine chemicals described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example having as the active polypeptide of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 7th row, 82-88 and 463-467 capable shown in)) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of the raising palmitinic acid described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give the raising palmitinic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide (for example having) as the active polypeptide of protein or its homologue shown in Table II the 3rd row, 82-88 and 463-467 are capable (for example as Table II the 7th row, 82-88 and 463-467 are capable shown in), perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give the raising palmitinic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 7th row, 82-88 and 463-467 capable shown in);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give the raising palmitinic acid that has described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 7th row, 82-88 and 463-467 capable shown in);

(f) express the transgenosis of coded protein, described protein give the raising palmitinic acid that has described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 7th row, 82-88 and 463-467 capable shown in);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have the raising palmitinic acid described in the literary composition active, by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method (for example having) as the active polypeptide of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 7th row, 82-88 and 463-467 capable shown in);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide or the used polypeptide of the inventive method (for example having as the active polypeptide of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 7th row, 82-88 and 463-467 capable shown in)).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.8.8] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example after active expression of polypeptides of protein shown in Table II the 3rd row, 82-88 and 463-467 are capable or its homologue (for example Table II the 5th or 7 row, 82-88 and 463-467 capable shown in polypeptide) or activity, give palmitinic acid and increase improving coded protein or have.

[0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.8] to [0067.0.0.8] saw for disclosing of [0055.0.0.8] to [0067.0.0.8] these paragraphs

Above [0068.0.5.8] and [0069.0.5.8] sees for disclosing of [0068.0.5.8] and [0069.0.5.8] these paragraphs [0068.0.0.0] and [0069.0.0.0]

Above [0070.0.6.8] and [0071.0.5.8] sees for disclosing of [0070.0.6.8] and [0071.0.5.8] these paragraphs [0070.0.5.5] and [0071.0.0.0]

[0072.0.8.8] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has other compound, for example palmitinic acid, Zoomeric acid, stearic acid, oleic acid, alpha-linolenic acid and/or linolic acid except palmitinic acid, the triacylglycerol that contains palmitinic acid, lipid, oils and/or fat.

[0073.0.8.8] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of protein with polypeptide of the present invention or the used polypeptide active of the inventive method or its homologue (for example Table II the 5th or 7 row, 82-88 and 463-467 capable shown in) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of promptly giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and other optional free and/or conjugated fatty acid.

[0074.0.5.8] sees [0074.0.5.5] for disclosing of this paragraph

[0075.0.0.0] was to [0084.0.0.0] above [0075.0.0.8] to [0084.0.0.8] saw for disclosing of [0075.0.0.8] to [0084.0.0.8] these paragraphs

[0085.0.8.8] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I the 5th and 7 row, 82-88 and 463-467 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as Table I the 5th or 7 row, 82-88 and nucleotide sequence or derivatives thereof shown in 463-467 is capable, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

Above [0086.0.0.8] and [0087.0.0.8] sees for disclosing of [0086.0.0.8] and [0087.0.0.8] these paragraphs [0086.0.0.0] and [0087.0.0.0]

[0088.0.8.8] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified fatty acid content.Because the nutritive value that for example is used for the plant of poultry depends on above-mentioned indispensable fatty acid and as the big scale of construction of the lipid acid in fodder energy source, so this is very important for the plant breeder.Improve or produced as Table II the 5th or 7 row, 82-88 and 463-467 capable shown in after the activity of polypeptide, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, thus the transgenic plant that produced grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0088.1.0.8], [0089.0.0.8], [0090.0.0.8] and [0091.0.5.8] is for [0088.1.0.8], [0089.0.0.8], top [0088.1.0.0] seen in disclosing of [0090.0.0.8] and [0091.0.5.8] these paragraphs, [0089.0.0.0], [0090.0.0.0] and [0091.0.0.0]

[0092.0.0.0] was to [0094.0.0.0] above [0092.0.0.8] to [0094.0.0.8] saw for disclosing of [0092.0.0.8] to [0094.0.0.8] these paragraphs

[0095.0.5.8], [0096.0.5.8] and [0097.0.5.8] are for [0095.0.5.8], and top [0095.0.5.5], [0096.0.5.5] and [0097.0.0.0] are seen in disclosing of [0096.0.5.8] and [0097.0.5.8] these paragraphs

[0098.0.8.8] in preferred embodiments, fine chemicals (palmitinic acid) be produce according to the present invention and carry out isolating where necessary.It is favourable as stearic acid, Zoomeric acid, oleic acid, alpha-linolenic acid and/or linolic acid mixture or other fatty acid mixt that the method according to this invention produces other lipid acid.

Above [0099.0.5.8] and [0100.0.5.8] sees for disclosing of [0099.0.5.8] and [0100.0.5.8] these paragraphs [0099.0.5.5] and [0100.0.5.5]

Above [0101.0.5.8] and [0102.0.5.8] sees for disclosing of [0101.0.5.8] and [0102.0.5.8] these paragraphs [0101.0.0.0] and [0102.0.5.5]

[0103.0.8.8] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as the sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as the sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer as sequence shown in Table III the 7th row, 82-88 and 463-467 are capable obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as the consensus sequence of sequence shown in Table IV the 7th row, 82-88 and 463-467 are capable and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, 82-88 and 463-467 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

Above [0103.1.0.8] and [0103.2.0.8] sees for disclosing of [0103.1.0.8] and [0103.2.0.8] these paragraphs [0103.1.0.0] and [0103.2.0.0]

[0104.0.8.8] in one embodiment, sequence had one or more Nucleotide different shown in sequence, preferred Table I A the 5th or 7 row, 82-88 and 463-467 shown in the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 82-88 and 463-467 are capable were capable, and not by Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I A the 5th or 7 row, 82-88 and 463-467 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 82-88 and 463-467 capable shown in sequence, preferred Table I A the 5th or 7 row, 82-88 and 463-467 capable shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 82-88 and 463-467 capable shown in sequence, preferred Table I A the 5th or 7 row, 82-88 and 463-467 capable shown in polypeptide of sequence.

Above [0105.0.0.8] to [0107.0.0.8] sees for disclosing of [0105.0.0.8] to [0107.0.0.8] these paragraphs [0105.0.0.0] and [0107.0.0.0]

Advantageously improved in [0108.0.8.8] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in 82-88 and 463-467 are capable, from Table II the 5th or 7 row, aminoacid sequence shown in 82-88 and 463-467 are capable is derived or is listed as from containing Table IV the 7th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 82-88 and 463-467 are capable, perhaps its coding has as Table II the 5th or 7 row, protein enzymic activity shown in 82-88 and 463-467 are capable or bioactive polypeptide or for example give derivative or the homologue that the fine chemicals linolic acid increases behind its expression or active the increasing.

[0109.0.5.8] sees [0109.0.0.0] for disclosing of this paragraph

[0110.0.8.8] helps the nucleic acid molecule of the polypeptide that the inventive method and coding have the used polypeptide active of the used or of the present invention process of the inventive method (for example as protein shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable or by as the protein of nucleic acid molecule encoding shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable or its homologue (shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable)) and can determine from generally open database.

[0111.0.0.8] sees [0111.0.0.0] for disclosing of this paragraph

The nucleic acid molecule that uses in [0112.0.8.8] the inventive method is the isolated nucleic acid sequences form, its coding has as the polypeptide of polypeptide active shown in Table II the 3rd row, 82-88 and 463-467 are capable or has polypeptide as peptide sequence shown in Table II the 5th and 7 row, 82-88 and 463-467 are capable, and gives each fine chemicals and increase.

Above [0113.0.0.8] to [0120.0.0.8] sees for disclosing of [0113.0.0.8] to [0120.0.0.8] these paragraphs [0113.0.0.0] and [0120.0.0.0]

[0121.0.8.8] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with the difference that one or more amino acid moleculars are arranged as peptide sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable or its functional homologue as herein described, described artificial sequence is preferably given aforementioned activity, and promptly giving each fine chemicals after improving its activity increases.

Above [0122.0.0.8] to [0127.0.0.8] sees for disclosing of [0122.0.0.8] to [0127.0.0.8] these paragraphs [0122.0.0.0] and [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.8.8] polymerase chain reaction (PCR) amplification is used (for example as the primer shown in Table III the 7th row, 82-88 and 463-467 are capable) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 82-88 and 463-467 capable shown in sequence or produce as sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable.

[0129.0.8.8] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence was from described comparison shown in Table IV the 7th row, 82-88 and 463-467 were capable.

[0130.0.8.8] sees [0130.0.0.0] for disclosing of this paragraph.

[0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.8] to [0138.0.0.8] saw for disclosing of [0131.0.0.8] to [0138.0.0.8] these paragraphs

[0139.0.8.8] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 5th or 7 row, 82-88 and 463-467 capable shown in sequence hybridization, and coding is expressed and is had the active peptide of the palmitinic acid of increasing.

Above [0140.0.0.8] to [0146.0.0.8] sees for disclosing of [0140.0.0.8] to [0146.0.0.8] these paragraphs [0140.0.0.0] and [0146.0.0.0]

[0147.0.8.8] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 5th or 7 row, 82-88 and 463-467 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.8.8] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 82-88 and 463-467 are capable, preferred Table I B the 5th or 7 row, nucleotide sequence shown in 82-88 and 463-467 are capable or its funtion part homology are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases fine chemicals.

[0149.0.8.8] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 5th or 7 row, 82-88 and 463-467 capable shown in one of nucleotide sequence or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (increasing as giving each fine chemicals) and as Table II the 5th or 7 row, 82-88 with 463-467 is capable, preferred Table II B the 5th or 7 row, 82-88 and the 463-467 protein shown in capable.

[00149.1.0.8] randomly, with Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 5th or 7 row, 82-88 and 463-467 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity is for being known activity or being used for these protein of note as the protein shown in Table II the 3rd row, 82-88 and 463-467 are capable.

[0150.0.8.8] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 5th or 7 row, 82-88 and 463-467 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving methionine(Met) when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 82-88 and 463-467 capable shown in sense strand, Table I the 5th or 7 row, 82-88 and the 463-467 of one of sequence capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 82-88 and 463-467 capable shown in the right PCR of primer will produce as Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 7th is listed as, 82-88 and 463-467 capable shown in the fragment of polynucleotide sequence.

[0151.0.0.8] sees [0151.0.0.0] for disclosing of this paragraph

[0152.0.8.8] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as the abundant homology of aminoacid sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable, make this albumen or its part keep to participate in produce the ability of fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising palmitinic acid as be shown in the examples.

[0153.0.8.8] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, has activity as protein or its part shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable as polypeptide shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable.

[0154.0.8.8] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

Above [0155.0.0.8] and [0156.0.0.8] sees for disclosing of [0155.0.0.8] and [0156.0.0.8] these paragraphs [0155.0.0.0] and [0156.0.0.0]

[0157.0.8.8] the present invention relate in addition owing to the genetic code degeneracy be different from Table I the 5th or 7 row, 82-88 and 463-467 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase) for example comprises as the polypeptide of consensus sequence shown in Table IV the 7th row, 82-88 and 463-467 are capable or as the nucleic acid molecule of polypeptide shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable or its function homologue.Preferably, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as the consensus sequence shown in Table IV the 7th row, 82-88 and 463-467 are capable or as polypeptide or its function homologue shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as the consensus sequence shown in Table IV the 7th row, 82-88 and 463-467 are capable or as the basic homology of aminoacid sequence of polypeptide shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I A the 5th or 7 row, 82-88 and the 463-467 sequence shown in capable.Preferably, nucleic acid molecule of the present invention is a functional homologous compound or identical with nucleic acid molecule shown in Table I B the 5th or 7 row, 82-88 and 463-467 are capable.

[0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.8] to [0160.0.0.8] saw for disclosing of [0158.0.0.8] to [0160.0.0.8] these paragraphs

[0161.0.8.8] therefore, in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor length are at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 82-88 and 463-467 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.8] sees [0162.0.0.0] for disclosing of this paragraph

[0163.0.8.8] preferably, under stringent condition with Table I the 5th or 7 row, 82-88 and 463-467 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.8] sees [0164.0.0.0] for disclosing of this paragraph

[0165.0.8.8] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 82-88 and 463-467 capable shown in sequence).

Above [0166.0.0.8] and [0167.0.0.8] sees for disclosing of [0166.0.0.8] and [0167.0.0.8] these paragraphs [0166.0.0.0] and [0167.0.0.0]

[0168.0.8.8] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are with different as the contained sequence of sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable, but have kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the aminoacid sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding is with identical at least about 60% as the sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable, more preferably with identical at least about 70% as one of sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable, even more preferably with as the sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable at least about 80%, 90% or 95% homology, and most preferably with identical at least about 96%, 97%, 98% or 99% as the sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table II B the 7th row, 82-88 and 463-467 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 82-88 with 463-467 is capable, preferred Table II B the 7th row, 82-88 and the 463-467 aminoacid sequence shown in capable is at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 82-88 and 463-467 are capable, preferred Table II B the 7th row, 82-88 is identical at least about 60% with the sequence shown in 463-467 is capable, more preferably with as Table II the 5th or 7 be listed as, 82-88 and 463-467 are capable, preferred Table II B the 7th row, 82-88 is identical at least about 70% with one of sequence shown in 463-467 is capable, even more preferably with as Table II the 5th or 7 be listed as, 82-88 and 463-467 are capable, preferred Table II B the 7th row, sequence shown in 82-88 and 463-467 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 82-88 and 463-467 are capable, preferred Table II B the 7th row, sequence shown in 82-88 and 463-467 are capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.0] was to [0175.0.0.0] above [0169.0.0.8] to [0175.0.5.8] saw for disclosing of [0169.0.0.8] to [0175.0.5.8] these paragraphs

[0176.0.8.8] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in 82-88 and 463-467 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 82-88 and 463-467 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in 82-88 and 463-467 are capable has essentially identical character and discerns.

[0177.0.8.8] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the functional equivalent that nucleotide sequence obtained shown in 82-88 and 463-467 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 82-88 and 463-467 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in 82-88 and 463-467 are capable.

[0178.0.0.8] sees [0178.0.0.0] for disclosing of this paragraph

[0179.0.8.8] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I the 5th or 7 row, 82-88 and 463-467 capable shown in), and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table II B the 7th is listed as, 82-88 and 463-467 capable shown in the nucleic acid molecule of homologous protein of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to encoding sequence, in sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable, introduce sudden change.

[0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.8] to [0183.0.0.8] saw for disclosing of [0180.0.0.8] to [0183.0.0.8] these paragraphs

[0184.0.8.8] is employed to have as Table I the 5th or 7 row, 82-88 and 463-467 are capable, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in 82-88 and 463-467 are capable, perhaps come Table II the 5th or 7 row freely, 82-88 and 463-467 are capable, preferred Table II B the 7th row, the homologous compound of the nucleotide sequence of sequence shown in 82-88 and 463-467 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.8.8] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I the 5th or 7 row, 82-88 with 463-467 is capable, preferred Table I B the 7th row, 82-88 and the 463-467 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 82-88 with 463-467 is capable, other nucleotide sequence of not showing in preferred Table I B the 7th row, 82-88 and the 463-467 arbitrary sequence shown in capable.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 82-88 with 463-467 is capable, preferred Table I B the 7th row, 82-88 and the 463-467 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.8.8] also preferred the inventive method comprise as Table II the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table II B the 7th row, 82-88 and 463-467 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table II B the 7th row, 82-88 and 463-467 capable shown in sequence identical.

[0187.0.8.8] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table II B the 7th row, 82-88 and 463-467 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table II B the 7th row, 82-88 and 463-467 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.8.8] still has the basic biologic activity of polypeptide of the present invention (being its active basic reduction that do not have) of giving fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active comparing with polypeptide expressed activity as Table II the 5th or 7 row, 82-88 and shown in 463-467 is capable and under the same conditions do not reduced substantially.In one embodiment, polypeptide of the present invention is to comprise as sequence shown in Table II B the 7th row, 82-88 and 463-467 are capable or by its homologue of forming.

[0189.0.8.8] as the homologous compound of sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.8], [0191.0.5.8], [00191.1.0.8] and [0192.0.0.8] to [0203.0.0.8] is for [0190.0.0.8], [0191.0.5.8], [0191.1.0.8] and [0192.0.0.8] is above disclosing of [0203.0.0.8] these paragraphs seen [0190.0.0.0], [0191.0.5.5], [0191.1.0.0] and [0192.0.0.0] is to [0203.0.0.0]

[0204.0.8.8] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table II B the 7th row, 82-88 and 463-467 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given fine chemicals quantity in biological or its part increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 7th row, 82-88 and 463-467 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as the primer shown in Table III the 7th row, 82-88 and 463-467 are capable or primer amplification from cDNA library or genomic library is obtained by using, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 82-88 and 463-467 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described peptide coding is as Table II the 5th or 7 row, 82-88 and 463-467 is capable, the structural domain of preferred Table II B the 7th row, 82-88 and the 463-467 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, nucleic acid molecule shown in 82-88 and 463-467 are capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 82-88 and 463-467 are capable

The fragment of preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt; Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A or IB the 5th or 7 row, 82-88 and 463-467 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule do not form by Table I A or IB the 5th or 7 row, 82-88 and 463-467 by the sequence shown in capable.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 82-88 and 463-467 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as the peptide sequence shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as the protein of sequence shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable at least 30% identical and with Table II A or IIB the 5th or 7 row, 82-88 and 463-467 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.0] was to [0226.0.0.0] above [0205.0.0.8] to [0226.0.0.8] saw for disclosing of [0205.0.0.8] to [0226.0.0.8] these paragraphs

[0227.0.8.8] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 82-88 and the capable sequence or derivatives thereof of mentioning of 463-467, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of palmitinic acid, Zoomeric acid, stearic acid and/or oleic acid biosynthetic pathway of at least one lipid acid of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 82-88 and 463-467 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.0] was to [0230.0.0.0] above [0228.0.5.8] to [0230.0.5.8] saw for disclosing of [0228.0.5.8] to [0230.0.5.8] these paragraphs

[0231.0.8.8] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened the degraded palmitinic acid simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.8] to [0276.0.0.8] saw for disclosing of [0232.0.0.8] to [0276.0.0.8] these paragraphs

[0277.0.5.8] sees [0277.0.5.5] for disclosing of this paragraph

[0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.8] to [0282.0.0.8] saw for disclosing of [0278.0.0.8] to [0282.0.0.8] these paragraphs

[0283.0.8.8] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-antibody as polypeptide shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable or its antigen part, polypeptide (polypeptide for example of the present invention or its fragment) generation that it can comprise above-mentioned sequence or be made up of above-mentioned sequence by the standard technique utilization.Preferably specificity is in conjunction with the monoclonal antibody as polypeptide shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable.

[0284.0.0.8] sees [0284.0.0.0] for disclosing of this paragraph

[0285.0.8.8] the present invention relates to have as sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable in one embodiment.

[0286.0.8.8] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in 82-88 and 463-467 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in 82-88 and 463-467 are capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to and comprising more than a polypeptide as consensus sequence shown in Table IV the 7th row, 82-88 and 463-467 are capable.

[0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.8] to [0290.0.0.8] saw for disclosing of [0287.0.0.8] to [0290.0.0.8] these paragraphs

[0291.0.8.8] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise and contain plant or microorganism specificity consensus sequence or by its polypeptide of forming.In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in 82-88 and 463-467 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in 82-88 and 463-467 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable.

[0292.0.0.8] sees [0292.0.0.0] for disclosing of this paragraph

[0293.0.8.8] the present invention relates to give the polypeptide that each fine chemicals increases in one embodiment in biological or its part, described polypeptide is by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention coding.

In one embodiment, polypeptide of the present invention have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help to form as the sequence of nucleic acid molecule encoding shown in Table I A or IB the 5th or 7 row, 82-88 and 463-467 are capable.

[0294.0.8.8] in one embodiment, the present invention relates to have as Table II the 3rd row, the polypeptide of activity of proteins shown in 82-88 and 463-467 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in 82-88 and 463-467 are capable.

[0295.0.0.8], [0296.0.0.8] and [0297.0.5.8] are for [0295.0.0.8], and disclosing of [0296.0.0.8] and [0297.0.5.8] these paragraphs sees that top [0295.0.0.0] is to [0297.0.0.0]

The chemical of [00297.1.0.8] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 82-88 and 463-467 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.

[0298.0.8.8] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with the abundant homologous aminoacid sequence of aminoacid sequence shown in Table II the 5th or 7 row, 82-88 and the 463-467, thereby this protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable.

[0299.0.8.8] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of aminoacid sequence shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable.

[0300.0.8.8] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,82-88 and 463-467 capable shown in sequence different.Therefore, this polypeptide contains with complete amino acid sequence homology as sequence shown in Table II A or IIB the 5th or 7 row, 82-88 and 463-467 are capable and is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.8] sees [0301.0.0.0] for disclosing of this paragraph

The biologically-active moiety of [0302.0.8.8] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence of the aminoacid sequence shown in 82-88 and 463-467 are capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.8] sees [0303.0.0.0] for disclosing of this paragraph

[0304.0.8.8] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as the active of polypeptide shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.5.8], [0306.0.5.8] and [0306.1.0.8] are for [0305.0.5.8], and top [0305.0.0.0], [0306.0.0.0] and [0306.1.0.0] are seen in disclosing of [0306.0.5.8] and [0306.1.0.8] these paragraphs

Above [0307.0.0.8] and [0308.0.0.8] sees for disclosing of [0307.0.0.8] and [0308.0.0.8] these paragraphs [0307.0.0.0and[0308.0.0.0]

[0309.0.8.8] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned, as Table II the 5th or 7 row, be meant polypeptide shown in 82-88 and 463-467 are capable with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " non-polypeptide " during 82-88 and 463-467 are capable or " other polypeptide " are meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with the homology not basically of the polypeptide with protein active, for example do not give described in the literary composition active and from the protein of identical or different biology.

[0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.8] to [0334.0.0.8] saw for disclosing of [0310.0.0.8] to [0334.0.0.8] these paragraphs

[0335.0.8.8] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of one of sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.8] to [0342.0.0.8] saw for disclosing of [0336.0.0.8] to [0342.0.0.8] these paragraphs

[0343.0.8.8] as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 82-88 and 463-467 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as one of sequence shown in Table I the 5th or 7 row, 82-88 and 463-467 are capable or its homologue in a kind of organism.

[0344.0.0.8] is to [0350.0.0.8], [0351.0.5.8] and [0352.0.0.8] to [0361.0.0.8] for [0344.0.0.8] to [0350.0.0.8], [0351.0.5.8] and [0352.0.0.8] above disclosing of [0361.0.0.8] these paragraphs seen [0344.0.0.0] to [0361.0.0.0]

[0362.0.8.8] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or each fine chemicals of its part increases) carry out genetically modified any cell, the nucleic acid molecule of described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide (for example coding has the polypeptide of protein as polypeptide active shown in Table II the 3rd row, 82-88 and 463-467 are capable).Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, cytoactive improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Transgenosis with the active polypeptide of protein (as polypeptide shown in Table II the 3rd row, 82-88 and 463-467 are capable) is meant in the text because genomic regulation and control or manipulation, improves having protein (as Table II the 3rd row, 82-88 and polypeptide shown in 463-467 is capable) activity active or similar active polypeptide in cell or biological or its part.Example and the inventive method are described in above.

[0363.0.0.8], [0364.0.5.8] and [0365.0.0.8] to [0379.0.5.8] be for [0363.0.0.8], [0364.0.5.8] and [0365.0.0.8] above disclosing of [0379.0.5.8] these paragraphs seen [0363.0.0.0] to [0379.0.0.0]

[0380.0.5.8], [0381.0.0.8] and [0382.0.0.8] is for [0380.0.5.8], top [0380.0.5.5] seen in disclosing of [0381.0.0.8] and [0382.0.0.8] these paragraphs, [0381.0.0.0] and [0382.0.0.0] [0383.0.5.8], [0384.0.0.8], [0385.0.5.8] and [0386.0.5.8] is for [0383.0.5.8], [0384.0.0.8], top [0383.0.5.5] seen in disclosing of [0385.0.5.8] and [0386.0.5.8] these paragraphs, [0384.0.0.0], [0385.0.5.5] and [0386.0.5.5]

[0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.8] to [0392.0.0.8] saw for disclosing of [0387.0.0.8] to [0392.0.0.8] these paragraphs

[0393.0.8.8] the present invention relates to identify and gives the method that the compound that becomes more meticulous in the cell produces the gene product that increases that it comprises following step in one embodiment:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 82-88 and 463-467 is capable, preferred Table I B the 5th or 7 row, 82-88 and the 463-467 nucleic acid molecule shown in capable is hybridized under lax stringent condition nucleic acid molecule, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.0] was to [0416.0.0.0] above [0394.0.0.8] to [0415.0.0.8] and [0416.0.5.8] saw for disclosing of [0394.0.0.8] to [0415.0.0.8] and [0416.0.5.8] these paragraphs

[0417.0.5.8] and [0418.0.0.8] to [0430.0.0.8] for [0417.0.5.8] and [0418.0.0.8] above disclosing of [0430.0.0.8] these paragraphs seen [0417.0.5.5] and [0418.0.0.0] to [0430.0.0.0]

[0431.0.5.8], [0432.0.5.8], [0433.0.0.8] and [0434.0.0.8] be for [0431.0.5.8], and [0432.0.5.8], [0431.0.0.0] was to [0434.0.0.0] above the disclosing of [0433.0.0.8] and [0434.0.0.8] these paragraphs seen

[0435.0.5.5] was to [0440.0.5.5] above [0435.0.5.8] to [0440.0.5.8] saw for disclosing of [0435.0.5.8] to [0440.0.5.8] these paragraphs

Above [0441.0.0.8] and [0442.0.5.8] sees for disclosing of [0441.0.0.8] and [0442.0.5.8] these paragraphs [0441.0.0.0] and [0442.0.5.5]

[0443.0.0.8] sees [0443.0.0.0] for disclosing of this paragraph

Above [0444.0.5.8] and [0445.0.5.8] sees for disclosing of [0444.0.5.8] and [0445.0.5.8] these paragraphs [0444.0.5.5] and [0445.0.5.5]

[0446.0.0.0] was to [0453.0.0.0] above [0446.0.0.8] to [0453.0.0.8] saw for disclosing of [0446.0.0.8] to [0453.0.0.8] these paragraphs

Above [0454.0.5.8] and [0455.0.5.8] sees for disclosing of [0454.0.5.8] and [0455.0.5.8] these paragraphs [0454.0.5.5] and [0455.0.5.5]

[0456.0.0.8] sees [0456.0.0.0] for disclosing of this paragraph

[0457.0.5.5] was to [0460.0.5.5] above [0457.0.5.8] to [0460.0.5.8] saw for disclosing of [0457.0.5.8] to [0460.0.6.8] these paragraphs

[0461.0.8.8] embodiment 10: clone SEQ ID NO:7435 is used for expressing plant

[0462.0.0.8] sees [0462.0.0.0] for disclosing of this paragraph

[0463.0.8.8] passes through pcr amplification SEQ ID NO:7435 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.5.8], [0465.0.5.8] and [0466.0.0.8] are for [0464.0.5.8], and top [0464.0.5.5], [0465.0.5.5] and [0466.0.0.0] are seen in disclosing of [0465.0.5.8] and [0466.0.0.8] these paragraphs

[0467.0.8.8] selects following primer sequence for genes of SEQ ID NO:7435:

I) forward primer (SEQ ID NO:7569)

atggagacca?atttttcctt?cgact

Ii) reverse primer (SEQ ID NO:7570)

ctattgaaat?accggcttca?atattt

[0468.0.0.0] was to [0479.0.0.0] above [0468.0.0.8] to [0479.0.0.8] saw for disclosing of [0468.0.0.8] to [0479.0.0.8] these paragraphs

[0480.0.8.8] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:7435

[0481.0.0.8] sees [0481.0.0.0] for disclosing of this paragraph

[0482.0.0.0] was to [0513.0.0.0] above [0482.0.0.8] to [0513.0.0.8] saw for disclosing of [0482.0.0.8] to [0513.0.0.8] these paragraphs

[0514.0.8.8] selects as another kind of, as people such as Geigenberger (Plant Cell﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect amino acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??YDR513W	Palmitinic acid (C16:0)	??GC	??1.17	??1.74

??YDR447C	Palmitinic acid (C16:0)	??GC	??1.19	??2.81
					??YBR089C-A	Palmitinic acid (C16:0)	??GC	??1.55	??1.95
??b3256	Palmitinic acid (C16:0)	??GC	??1.15	??1.17
					??YGR126W	Palmitinic acid (C16:0)	??GC	??1.34	??2.67
??YPL099C	Palmitinic acid (C16:0)	??GC	??1.21	??1.55
					??b0399	Palmitinic acid (C16:0)	??GC	??1.20	??1.27
??b0849	Palmitinic acid (C16:0)	??GC	??1.17	??1.303
					??b3457	Palmitinic acid (C16:0)	??GC	??1.16	??1.36
??b3578	Palmitinic acid (C16:0)	??GC	??1.16	??1.271
					??b3644	Palmitinic acid (C16:0)	??GC	??1.16	??1.42
??b4129	Palmitinic acid (C16:0)	??GC	??1.33	??1.36

[0515.0.5.8] the 2nd row have shown the lipid acid of being analyzed.The 4th row and the 5th row have shown the ratio of lipid acid between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

Above [0516.0.0.8] and [0517.0.5.8] sees for disclosing of [0516.0.0.8] and [0517.0.5.8] these paragraphs [0516.0.0.0] and [0517.0.0.0]

[0518.0.0.0] was to [0530.0.0.0] above [0518.0.0.8] to [0529.0.0.8] and [0530.0.5.8] saw for disclosing of [0518.0.0.8] to [0529.0.0.8] and [0530.0.5.8] these paragraphs

[0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.8] to [0530.6.0.8] saw for disclosing of [0530.1.0.8] to [0530.6.0.8] these paragraphs

[0531.0.0.0] was to [0534.0.0.0] above [0531.0.0.8] to [0533.0.0.8] and [0534.0.5.8] saw for disclosing of [0531.0.0.8] to [0533.0.0.8] and [0534.0.5.8] these paragraphs

[0535.0.0.0] was to [0538.0.0.0] above [0535.0.0.8] to [0537.0.0.8] and [0538.0.5.8] saw for disclosing of [0535.0.0.8] to [0537.0.0.8] and [0538.0.5.8] these paragraphs

[0539.0.0.0] was to [0543.0.0.0] above [0539.0.0.8] to [0542.0.0.8] and [0543.0.5.8] saw for disclosing of [0539.0.0.8] to [0542.0.0.8] and [0543.0.5.8] these paragraphs

Above [0544.0.0.8] to [0547.0.0.8] and [0548.0.5.8] see for disclosing of [0544.0.0.8] to [0547.0.0.8] and [0548.0.5.8] to [0552.0.0.8] these paragraphs to [0552.0.0.8]

[0544.0.0.0] is to [0552.0.0.0]

[0552.2.0.8] sees [0552.2.0.0] for disclosing of this paragraph

[0553.0.8.8]

1. produce the method for palmitinic acid, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation palmitinic acid.

2. produce the method for palmitinic acid, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable, and described nucleic acid molecule gives that palmitinic acid quantity increases in biology or its part;

B) contain nucleic acid molecule just like the nucleic acid molecule shown in Table I the 5th or 7 row, 82-88 and the 463-467;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of palmitinic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of palmitinic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that palmitinic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 82-88 and 463-467 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that palmitinic acid quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that palmitinic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 82-88 and 463-467 capable shown in consensus sequence polypeptide and give biology or its part in palmitinic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give palmitinic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded palmitinic acid.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded palmitinic acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 82-88 and 463-467 are capable, and described nucleic acid molecule gives that palmitinic acid quantity increases in biology or its part;

B) contain nucleic acid molecule just like the nucleic acid molecule shown in Table I the 5th or 7 row, 82-88 and the 463-467;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of palmitinic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of palmitinic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that palmitinic acid quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 82-88 and 463-467 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that palmitinic acid quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that palmitinic acid quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 82-88 and 463-467 capable shown in consensus sequence polypeptide and give biology or its part in palmitinic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give palmitinic acid quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A the 5th or 7 row, 82-88 and 463-467 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 82-88 and 463-467 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in palmitinic acid quantity increase, described method comprises:

(b) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part palmitinic acid quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(c) measure in cell, tissue, plant or the microorganism, perhaps palmitinic acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(d) relatively identify agonist or antagonist by the palmitinic acid level that will measure or expression of polypeptides level and standard palmitinic acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the palmitinic acid generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of palmitinic acid quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of palmitinic acid quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the palmitinic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that palmitinic acid increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce palmitinic acid;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) the palmitinic acid level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give palmitinic acid level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the palmitinic acid generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that palmitinic acid quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce palmitinic acid;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) the palmitinic acid level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give palmitinic acid level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that palmitinic acid increases after expression.

25. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control palmitinic acid level at biology.

26. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make the anti-palmitinic acid synthetic weedicide that suppresses of plant.

[0554.0.0.8] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.9] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.9] sees [0001.0.0.0]

[0002.0.9.9] because plant has plastid, so plant has some biosynthetic pathways, these approach are for being unique for the living organism the cyanobacteria.Nutrition is absolutely necessary and therefore is called as VITAMIN some plastid compounds for humans and animals.Nutraceutical two kinds must lipotropic component be provitamin A (β-Hu Luobusu) and vitamin-E.

Vitamin-E is classified by its pharmacotoxicological effect and chroman alcohol ring structure, rather than classifies according to its biosynthesizing.Vitamin-E comprises 8 kinds of solvable compositions of lipid of a class, and this constituents is subdivided into tocopherol and tocotrienols.Though tocopherol has the isoprenoid side chain from phytyl-PP, and the tocotrienols side chain is the derivative of geranyl geranyl-PP.The α of these subclass, β, γ and δ member are different from the degree that methylates in its 6-chroman alcohol ring structure.

Tocopherol group (1a-d) has saturated side chains, and tocotrienols group (2a-d) has unsaturated terminal chain:

1a, alpha-tocopherol: R ¹=R ²=R ³=CH ₃

1b, 5,8-dimethyl tocol: R ¹=R ³=CH ₃, R ²=H

1c, Gamma-Tocopherol: R ¹=H, R ²=R ³=CH ₃

1d ,-tocopherol: R ¹=R ²=H, R ³=CH ₃

2a, alpha-tocotrienol: R ¹=R ²=R ³=CH ₃

2b, β-tocotrienols: R ¹=R ³=CH ₃, R ²=H

2c, γ-tocotrienols: R ¹=H, R ²=R ³=CH ₃

2d ,-tocotrienols: R ¹=R ²=H, R ³=CH ₃

In the present invention, the vitamin-E meaning is meant all above-mentioned have active tocopherol of vitamin-E and tocotrienols.

The position of tocopherol α, β, γ and the δ methyl group of [0003.0.9.9] four class principal modes is different with number.Principal mode in the higher plant leaf is an alpha-tocopherol, and in seed the normally main isoform of Gamma-Tocopherol.In photosynthetic organism and living animal, and in isolated compound such as oils, tocopherol is mainly exercised antioxidant function.The antioxidant properties of tocopherol comes from the ability of its cancellation free radical, and different tocopherols can be the suitableeest antioxidant for different biosystems.For the mankind's animal use, it is active and related to multiple health field that alpha-tocopherol has homovitamin E, comprise have the preventing cardiovascular disease of being beneficial to, some cancer and cataract form.The amounts that realize the required vitamin-E of these effects are compared normally very high with feed rate 40 international unit every day (I.U.) of being advised, be 100-400 I.U., even up to 800I.U..In fat and oils, tocopherol prevents the unsaturated fatty acids acid oxidase.In these systems, Gamma-Tocopherol shows to have bigger effect.In fact, tocopherol is contained in the finished oils usually, to help stabilised fat acid.For the utilization of human health and food and feed, the plant with tocopherol content of raising is desired, is accompanied by tocopherol content and improves that the tocopherol composition obtains specialization in these plants.

[0004.0.9.9] tocopherol contains from the aromatic series head group of homogentisic acid (HGA) with from phytyl bisphosphate (the hydrocarbon polymer part of phytyl-DP).HGA is from shikimic acid pathway and phytyl-DP is the group that is concentrated and formed by 4 isoprenoid unit.Biosynthetic effect is considered to be mainly derived from plastid methyl-erythritol phosphoric acid approach to isoprenoid to tocopherol, rather than derives from the cytosol mevalonate pathway.First committed step in the tocopherol biosynthesizing, HGA and phytyl-DP concentrate and form 2-methyl-6-phytyl plastomer chinol, are the amylene transferring enzyme reactions of being undertaken by homogentisic acid phytyl transferring enzyme (HPT).Cyclisation afterwards and methylation reaction cause forming the main tocopherol of 4 classes.Enzymatic reaction in the tocopherol biosynthesizing is to identify before the 15-20, but the clone of the gene of encoding such enzymes only just carries out in recent years.

The biosynthesizing of [0005.0.9.9] tocopherol betides plastid and required enzyme is relevant with chloroplast envelope.The film dependency of enzyme makes and is difficult to purifying.Along with the arriving in genome epoch and providing of large number of biological (comprising that cytoalgae (Synechocystis sp.) PCC 6803 and mouse ear mustard belong to) whole genome sequence, might use the extra gene in bioinformatics technique evaluation and the clone's tocopherol pathway.

First enzyme in the tocopherol pathway of being cloned, gama-tocopherol methyl transferase (γ-TMT), utilize information biology in cytoalgae PCC 6803 and mouse ear mustard genus, to identify.In this research, display change seed tocopherol component when mouse ear mustard genus γ-TMT crosses expression in mouse ear mustard belongs to.Normal Gamma-Tocopherol for main tocopherol isomer in the mouse ear mustard genus seed almost all is converted into alpha-tocopherol.

First committed step in the HPT catalysis tocopherol pathway, and before do not identified.Found the slr1736 of HPT among the coding cytoalgae PCC 6803 when carrying out this research and identified that mouse ear mustard belongs to HTP.

There are the amylene transferring enzyme of catalysis isopentene group group and the condensation of allyl group chain and the amylene transferring enzyme of catalysis isopentene group chain and aromatic group condensation.The amylene transferring enzyme of catalysis isopentenylpyrophosphate and the condensation of allyl group chain subsequently has common feature (comprising the motif that is rich in Asp) and causes having the formation of 2 unitary compounds of isoprenoid, as geranylpyrophosphate or longer molecule, as rubber (it comprises more than 1,000 isoprenoid unit).The motif that is rich in Asp of the amylene transferring enzyme of catalysis and the condensation of non-isoprenoid group is different from the motif that is rich in Asp of allylic amylene transferring enzyme, and comprises the chlorophyll synthase that isopentene group is connected to the UbiA of 4 hydroxybutyric acid and isopentene group is connected to chlorophyllin fat.

First committed step in the biosynthesizing of [0006.0.9.9] tocopherol is that the phytyl chain transfer is catalytic to the fragrant amylene transferring enzyme of HGA.

Tocopherol, tocotrienols and plastoquinone can be classified as one group by head group classification, for quinone with anti-oxidation characteristics and with homogentisic acid as precursor.Plastoquinone is the important component in the quinones in the photosynthetic electron transport chain of plastid, and it also participates in provitamin A (β-Hu Luobusu; NorrisSR, (1995) .Plant Cell 7, biosynthesizing 2139-2149).

[0007.0.9.9] vitamin-E is mainly sent by feeding plant oil.Relevant antioxidant for clearing agent has important effect to vitamin-E as film.In the past few years, proposed again vitamin-E as several other functions of anti-hypercholesterolemiccompounds reagent and immunostimulant among the mankind (Beharka (1 997) .Methods Enzymol.282,247-263).

These have the active compound of vitamin-E is important neutral fat soluble antioxidant.Vitamin E deficiency causes the physiopathology situation in humans and animals.Therefore, in food and feed part, in pharmaceutical preparation and in the cosmetic applications, the vitamin-E compound is the additive of high economic worth.

[0008.0.9.9] in plant plastid, many isoprenoid approach are localized, and they are interrelated by its substrate, end product and regulation and control.These isoprenoid approach are for example monoterpene, diterpene, gibberic acid (giberillic acid), dormin, chlorophyll, phylloquinone, carotenoid, tocopherol, tocotrienols and plastoquinone biosynthesizing.In whole these approach, the amylene transferring enzyme participates in the biosynthesizing of these compounds.Consider the length of their side chains, diterpene, chlorophyll, phylloquinone, tocopherol and tocotrienols can be classified as the C of isoprenoid ₂₀-group.Another kind of classification by side chain desaturation degree can be classified as the phytyl group with for example chlorophyll, phylloquinone and tocopherol, and for example diterpene, tocotrienols, plastoquinone and carotenoid are classified as desaturation isoprenoid compounds group.

[0009.0.9.9] therefore, it is very important being used to produce the method for economy of vitamin-E or its precursor and food and the feed with content of vitamin E of raising.Jing Ji method is to utilize vitamin-E to produce biological biotechnological means especially, and these vitamin-E production biologies can be natural or pass through the genetic modification optimization.

Therefore, will need provide new enzymic activity or direct or indirect instrumentality for a long time, and therefore need have the alternative approach of advantageous feature, so that for example produce vitamin-E or its precursor in the genetically modified organism at biology.

Known the trial realizes that by cross expression phytyl/amylene transferase gene in genetically modified organism thereby the metabolite flow improves tocopherol and/or tocotrienols content; WO 00/63391, WO 00/68393, WO 01/62781 and WO 02/33060.

[0010.0.9.9] therefore, the quality that improves food and animal-feed is the vital task of food and fodder industry.This is inevitable, and is limited because for example be present in the vitamin-E of plant and certain micro-organisms for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is the spectrum of the VITAMIN in the balanced diet as far as possible, though a large amount of excessive specific concentrations that surpass in the food of some VITAMIN, only have some or seldom or do not have a positive effect.Further improve the quality unique possible be by adding other limited VITAMIN.

[0011.0.9.9] must add one or more VITAMIN to be fit to biological balance mode for the quality of food and animal-feed.

[0012.0.9.9] therefore, still exist great demand new and more suitable gene, described genes encoding participates in the biosynthetic enzyme of VITAMIN, particularly vitamin-E and can produce them and not form unnecessary byproduct in the particular industry scale.In the gene of selecting the biosynthesizing instrumentality, above-mentioned two features are particularly importants.On the one hand, but still need to obtain the development of the highest intrinsic energy VITAMIN such as vitamin-E, on the other hand, produce byproduct in process of production as few as possible.

[0013.0.0.9] sees [0013.0.0.0]

[0014.0.9.9] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is a vitamin-E.Therefore, in the present invention, term " fine chemicals " is meant " vitamin-E " as used herein.In addition, in another embodiment, term " fine chemicals " also refers to comprise the fine chemicals composition of vitamin-E as used herein.

[0015.0.9.9] in one embodiment, term " vitamin-E " or " fine chemicals " or " each fine chemicals " meaning are meant at least a active chemical compound of vitamin-E that has, and it is selected from " alpha-tocopherol, 5,8-dimethyl tocol, Gamma-Tocopherol, Delta-Tocopherol, alpha-tocotrienol, β-tocotrienols, γ-tocotrienols and δ-tocotrienols ".In preferred embodiments, term " fine chemicals " or term " vitamin-E " or term " each fine chemicals " meaning are meant at least a active chemical compound of vitamin-E that has, and it is selected from " alpha-tocopherol, 5,8-dimethyl tocol and Gamma-Tocopherol ".

Usually, the increase of the content of vitamin E meaning is meant that total content of vitamin E increases.Yet the increase of content of vitamin E also refers to above-mentioned change with the active 8 kinds of compounds contents of vitamin-E, must increase and need not total content of vitamin E.In the embodiment preferred, term " fine chemicals " meaning is meant the vitamin-E of free form or its salt or ester-formin or combining form.In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning is meant at least a active chemical compound of above-mentioned fine chemicals that has.

[0016.0.9.9] therefore the present invention relates to produce the method for vitamin-E, and it comprises:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins:

-YFL019C, YPL268W, YFL013C, b1829 and/or b1827 protein;

-b0161, b0970, b3160, b4063, b2699, b0112 and/or b1829 protein;

-b0785, b3938, YFL053W, b1827, b0986, b1829 and/or b0175 protein; And/or

-b0175, b0785, b0986, b3838, YFL053W and/or b1829 protein; With

(b) producing fine chemicals respectively in allowing described biology is vitamin-E, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol and/or vitamin-E precursor 2, makes biological growth under the condition of 3-dimethyl-5-phytyl chinol.

Particularly, the present invention relates to produce 2, the method for 3-dimethyl-5-phytyl chinol, alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol, it comprises:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins:

-for 2, YFL019C, YPL268W, YFL013C, b1829 and/or the b1827 protein of 3-dimethyl-5-phytyl quinone;

-for b0161, b0970, b3160, b4063, b2699, b0112 and/or the b1829 protein of alpha-tocopherol,

-for b0785, b3938, YFL053W, b1827, b0986, b1829 and/or the b0175 protein of 5,8-dimethyl tocol; And/or

-for b0175, b0785, b0986, b3838, YFL053W and/or the b1829 protein of Gamma-Tocopherol; With

(b) in allowing described biology, produce alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol or vitamin-E precursor 2 respectively, make biological growth under the condition of 3-dimethyl-5-phytyl chinol.

Therefore, the present invention relates to produce the method for vitamin-E, it comprises:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, 89-102 is capable or 472-482 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 the row, 89-102 is capable or 472-482 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) be vitamin-E, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol and/or vitamin-E precursor 2 allowing to produce fine chemicals respectively, make biological growth under the condition of 3-dimethyl-5-phytyl chinol.

Especially, the present invention relates to produce the method for vitamin-E, it comprises:

(a) improve in non-human being or one or more part or produce one or more following protein, described protein has for 2, Table II the 3rd row, 89-92 or 482 row of 3-dimethyl-5-phytyl chinol; Table II the 3rd row, 93-95 or 472-475 for alpha-tocopherol are capable; Table II the 3rd row, 96-100 or 476-477 for 5,8-dimethyl tocol are capable; And/or for Table II the 3rd row, 101-102 or the 478-481 of Gamma-Tocopherol capable shown in activity of proteins, for example have for 2 the Table I the 5th of 3-dimethyl-5-phytyl chinol or 7 row, 89-92 or 482 row; Table I the 5th or 7 row, 93-95 or 472-475 for alpha-tocopherol are capable; Table I the 5th or 7 row, 96-100 or 476-477 for 5,8-dimethyl tocol are capable; And/or for the Table I the 5th of Gamma-Tocopherol or 7 row, 101-102 or 478-481 capable shown in the sequence of the coded polypeptide of nucleic acid molecule; With

(b) allowing to produce fine chemicals, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol and/or vitamin-E precursor 2, make biological growth under the condition of 3-dimethyl-5-phytyl chinol.

[0016.1.9.9] therefore, term " fine chemicals " refers to and Table I-IV 89-92 or relevant " 2; 3-dimethyl-5-phytyl chinol " or its homologue of the 482 listed full sequences of row in one embodiment, and refer to " alpha-tocopherol " or its homologue relevant in one embodiment with I-IV 93-95 or the capable listed full sequence of 472-475, and refer to " 5,8-dimethyl tocol " or its homologue relevant in one embodiment, and refer to " Gamma-Tocopherol " or its homologue relevant in one embodiment with Table I-IV 101-102 or the capable listed full sequence of 478-481 with Table I 96-100 or the capable listed full sequence of 476-477.

In one embodiment, the inventive method is given more than one each fine chemicals, i.e. alpha-tocopherol, 5,8-dimethyl tocol, Gamma-Tocopherol or 2, and the content of 3-dimethyl-5-phytyl chinol increases.Therefore, in one embodiment, term " fine chemicals " refers to and Table I-IV the 91st, 94,100 or relevant " 2; 3-dimethyl-5-phytyl chinol ", " 5,8-dimethyl tocol " and " Gamma-Tocopherol " of the 102 listed full sequences of row, it is each fine chemicals that the inventive method is given more than one, promptly 2, the content of 3-dimethyl-5-phytyl chinol, alpha-tocopherol, 5,8-dimethyl tocol and Gamma-Tocopherol increases.Preferably, the inventive method gives 2, and 3-dimethyl-5-phytyl chinol, alpha-tocopherol, 5,8-dimethyl tocol and Gamma-Tocopherol content increase.In one embodiment, term " fine chemicals " refers to and Table I-IV the 92nd or relevant " 2; 3-dimethyl-5-phytyl chinol " and " 5,8-dimethyl tocol " of the 97 listed full sequences of row that promptly the inventive method gives 2, and 3-dimethyl-5-phytyl chinol and 5,8-dimethyl tocol content increase.In one embodiment, term " fine chemicals " refers to and Table I-IV the 96th, 98,99,101,476,477,478,479,480 or relevant " 5,8-dimethyl tocol " and " Gamma-Tocopherol " of the 481 listed full sequences of row.

Therefore, term " fine chemicals " can refer to " 2,3-dimethyl-5-phytyl chinol ", " alpha-tocopherol ", " 5,8-dimethyl tocol " and/or " Gamma-Tocopherol " according to environment and context.For the meaning of illustrating term " fine chemicals " is meant " 2,3-dimethyl-5-phytyl chinol ", " alpha-tocopherol ", " 5,8-dimethyl tocol " and/or " Gamma-Tocopherol ", also can use term " each fine chemicals ".

[0017.0.0.9] to [0018.0.0.9]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.9.9] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 89-102 and 472-482 are capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 89-102 and 472-482 were capable.

[0020.0.9.9] is surprisingly found out that, at least a yeast saccharomyces cerevisiae protein YFL019C, YPL268W and/or YFL013C and/or e. coli k12 protein b1829 and/or b1827 transgene expression in Arabidopis thaliana give 2, and 3-dimethyl-5-phytyl chinol (vitamin-E biosynthesizing precursor), particularly Gamma-Tocopherol and alpha-tocopherol increase.Therefore, the raising of this tocopherol biosynthesizing precursor level can help the generation of vitamin-E.For example, in one embodiment, 2,3-dimethyl-5-phytyl chinol level with to (the metabolism 2 of particularly encoding of biosynthetic other gene of vitamin-E, the gene of the enzyme of 3-dimethyl-5-phytyl chinol) expression is regulated and is enhanced together, so that produce vitamin-E and its precursor, for example 2,3-dimethyl-5-phytyl chinol cyclase and/or gama-tocopherol methyl transferase II or with coding Table II the 5th or 7 row, 89-102 or 472-482 capable shown in the gene of polypeptide.

Be surprisingly found out that, e. coli k12 protein b0161, b0970, b3160, b4063, b2699, b1829 and/or b0112 transgene expression in Arabidopis thaliana give the plant alpha-tocopherol that transforms increase.

Be surprisingly found out that, yeast saccharomyces cerevisiae protein YFL053W and/or e. coli k12 protein b0785, b3938, b1827, b1829, b0986 and/or b0175 transgene expression in Arabidopis thaliana give the plant 5,8-dimethyl tocol content that transforms increase.

Be surprisingly found out that, yeast saccharomyces cerevisiae protein YFL053W and/or e. coli k12 protein b0175, b0785, b03938, b0986, b1829 transgene expression in Arabidopis thaliana give the plant Gamma-Tocopherol content that transforms increase.

Therefore, be surprisingly found out that, e. coli k12 protein b1829 transgene expression in Arabidopis thaliana is given the plant that transforms 2, and 3-dimethyl-5-phytyl chinol, alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol (or each fine chemicals) content increase.Therefore, in one embodiment, described protein or its homologue are used to produce 2,3-dimethyl-5-phytyl chinol; In one embodiment, described protein or its homologue are used to produce alpha-tocopherol; In one embodiment, described protein or its homologue are used to produce 5,8-dimethyl tocol; In one embodiment, described protein or its homologue are used to produce Gamma-Tocopherol; In one embodiment, described protein or its homologue are used for producing and are selected from 2, one or more fine chemicals of 3-dimethyl-5-phytyl chinol, alpha-tocopherol, 5,8-dimethyl tocol and Gamma-Tocopherol.

Therefore, be surprisingly found out that e. coli k12 protein b1827 transgene expression in Arabidopis thaliana is given the plant that transforms 2,3-dimethyl-5-phytyl chinol and/or 5,8-dimethyl tocol (or each fine chemicals) content increases.Therefore, in one embodiment, described protein or its homologue are used to produce 2,3-dimethyl-5-phytyl chinol; In one embodiment, described protein or its homologue are used to produce 5,8-dimethyl tocol; In one embodiment, described protein or its homologue are used to produce 2,3-dimethyl-5-phytyl chinol and 5,8-dimethyl tocol.

Be surprisingly found out that, yeast saccharomyces cerevisiae protein YFL053W and Escherichia coli protein b0785, b3938, b0986 or b0175 transgene expression in Arabidopis thaliana give the plant 5,8-dimethyl tocol that transforms and/or Gamma-Tocopherol (or each fine chemicals) content increase.Therefore, in one embodiment, described protein or its homologue are used to produce 5,8-dimethyl tocol; In one embodiment, described protein or its homologue are used to produce Gamma-Tocopherol, and in one embodiment, described protein or its homologue are used to produce 5,8-dimethyl tocol and Gamma-Tocopherol.

[002 1.0.0.9] sees [002 1.0.0.0]

The sequence of [0022.0.9.9] e. coli k12 b1829 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the heat shock protein with protease activity.Therefore, in one embodiment, that the inventive method comprises is as shown here " coerce reaction; the pheromone reaction; the mating type decision; the protein of protein modification or have the active protein of proteolytic degradation or be sex-specific protein " purposes, proteolytic enzyme particularly, the heat shock protein that particularly has protease activity, the purposes of preferred heat shock protein htpX superfamily, it is used for for example by increasing vitamin-E precursor 2 as described, 3-dimethyl-5-phytyl chinol biosynthesizing produces each fine chemicals in biological or its part, be vitamin-E, alpha-tocopherol for example, 5,8-dimethyl tocol and/or Gamma-Tocopherol, preferred free or combining form.In another embodiment, the b1829 protein expression is enhanced with another gene of vitamin-E biosynthetic pathway, preferably another gene with coding participation vitamin-E generation (by intermediate product 2, the conversion of 3-dimethyl-5-phytyl chinol) is enhanced together.In one embodiment, in the methods of the invention, proteolytic enzyme, particularly have the heat shock protein of protease activity, the activity of preferred heat shock protein htpX superfamily is enhanced or produces, for example the activity from colibacillary enzyme or its homologue is enhanced or produces.

The sequence of e. coli k12 b1827 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be supposition have DNA in conjunction with the transcription repressor (IcIR family) in wing spirane structure territory, preferred acetate operon repressor superfamily.Therefore, in one embodiment, the inventive method comprise acetate operon repressor superfamily protein as shown here (preferably this proteinoid have the control of transcribing active, particularly have a supposition have the activity of DNA in conjunction with the transcription repressor (IcIR family) in wing spirane structure territory) purposes, it is used for for example by increasing vitamin-E precursor 2 as described, 3-dimethyl-5-phytyl chinol biosynthesizing produces each fine chemicals in biological or its part, be vitamin-E, for example 5,8-dimethyl tocol, preferably free or combining form.In another embodiment, the b1827 protein expression is enhanced with another gene of vitamin-E biosynthetic pathway, preferably participates in from 2 with coding, and another gene that 3-dimethyl-5-phytyl chinol produces vitamin-E is enhanced together.In one embodiment, in the methods of the invention, the DNA that has that described activity is for example supposed is enhanced or produces in conjunction with the activity of the transcription repressor (IcIR family) in wing spirane structure territory, the activity of preferred acetate operon repressor superfamily, for example being enhanced or producing from colibacillary this type of activity or its homologue.

The sequence of sequence b0785 e. coli k12 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be molybdenum cofactor biosynthesizing protein E.Therefore, in one embodiment, the inventive method comprises and as shown here " participates in VITAMIN, cofactor and prothetic group metabolism from colibacillary; Other melts combine; Protein bound; Molybdenum pterin bonded protein ", the purposes of preferred molybdenum pterin biosynthesizing protein E chain superfamily, preferred molybdenum cofactor biosynthesizing protein E or its homologue, as described its to be used for producing each fine chemicals in biological or its part be vitamin-E, particularly 5,8-dimethyl tocol and/or Gamma-Tocopherol, preferably free or combining form.In one embodiment, in the methods of the invention, the described activity for example activity of molybdenum cofactor biosynthesizing protein E is enhanced or produces, and for example the activity from colibacillary molybdenum cofactor biosynthesizing protein E or its homologue is enhanced or produces.

The sequence of e. coli k12 b3938 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the biosynthetic transcription repressor of methionine(Met).Therefore, in one embodiment, the inventive method comprises purposes from colibacillary metJ protein superfamily protein, the preferred biosynthetic transcription repressor of methionine(Met) or its homologue as shown here, as described its to be used for producing each fine chemicals in biological or its part be vitamin-E, particularly 5,8-dimethyl tocol and/or Gamma-Tocopherol, preferably free or combining form.In one embodiment, in the methods of the invention, the described activity for example activity of the biosynthetic transcription repressor of methionine(Met) is enhanced or produces, and for example the activity from the biosynthetic transcription repressor of colibacillary methionine(Met) or its homologue is enhanced or produces.

The sequence of e. coli k12 b0175 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be CDP-diester glycerine synthase.Therefore; in one embodiment; the inventive method comprises as shown here from colibacillary " participating in nucleotide metabolism; phosphatide biosynthesizing; lipid; lipid acid or isoprenoid metabolism; cell communication; the protein of signal transduction or cell sense and reaction "; preferred phosphatidic acid two cytidine acyltransferase superfamilies; the purposes of preferred CDP-diester glycerine synthase or its homologue; as described its to be used for producing each fine chemicals in biological or its part be vitamin-E; particularly 5,8-dimethyl tocol and/or Gamma-Tocopherol preferably dissociate or combining form.In one embodiment; in the methods of the invention; described activity is phosphatidic acid two cytidine acyltransferases for example; particularly the activity of CDP-diester glycerine synthase is enhanced or produces; for example from colibacillary phosphatidic acid two cytidine acyltransferases, particularly the activity of CDP-diester glycerine synthase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the DNA chain exchange with proteolytic enzyme and nuclease and the recombinant protein of recombinant protein recA superfamily.Therefore, in one embodiment, the inventive method comprises as shown here recombinating and the DNA repairing activity from the colibacillary DNA of having, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the protein that particularly has proteolytic enzyme and nuclease, the purposes of recombinant protein recA superfamily DNA chain exchange and recombinant protein or its homologue particularly, as described its to be used for producing each fine chemicals in biological or its part be vitamin-E, particularly increase alpha-tocopherol, the quantity of preferred free or combining form alpha-tocopherol.In one embodiment, in the methods of the invention, the exchange of DNA chain and recombinant protein, the particularly activity of recombinant protein recA superfamily that described activity for example has proteolytic enzyme and nuclease are enhanced or produce, and for example the activity from colibacillary these enzymes or its homologue is enhanced or produces.

The sequence of e. coli k12 b3160 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the monooxygenase with luciferase sample atpase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the monooxygenase with luciferase sample atpase activity of colibacillary supposition or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be alpha-tocopherol, particularly increase alpha-tocopherol, the preferred quantity of free or combining form alpha-tocopherol.In one embodiment, in the methods of the invention, the activity of the monooxygenase of supposing with luciferase sample atpase activity is enhanced or produces, and for example is enhanced or produces from the monooxygenase with luciferase sample atpase activity of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b0161 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease and/or heat shock protein.Therefore, in one embodiment, the inventive method comprises as shown here sending out from colibacillary extracellular protein/secretory protein or participation cell growth/form, proteolysis falls, protein bound, the protein of signal in the cell, preferred Helicobacterium serine protease superfamily protein, the purposes of preferred pericentral siphon serine protease (heat shock protein) or its homologue, it is used for to be alpha-tocopherol, particularly to increase alpha-tocopherol at biological or its part generation fine chemicals as described, the quantity of preferred free or combining form alpha-tocopherol.In one embodiment, in the methods of the invention, the activity of pericentral siphon serine protease (heat shock protein) is enhanced or produces, and for example the activity from colibacillary serine protease (heat shock protein) or its homologue is enhanced or produces.

The sequence of e. coli k12 b0970 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be possible glutamate receptor.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary apoptosis involvement (I type apoptosis), transmembrane signal transduction, passage/hole class transporter, the protein of unspecified signal transduction and/or protein bound (preferably from intestinal bacteria ybhL protein superfamily), the particularly possible glutamate receptor or the purposes of its homologue, as described its to be used for producing each fine chemicals in biological or its part be vitamin-E, particularly increase alpha-tocopherol, the quantity of preferred free or combining form alpha-tocopherol.In one embodiment, in the methods of the invention, the activity of described activity such as possible glutamate receptor (particularly intestinal bacteria ybhL protein superfamily) is enhanced or produces, and for example is enhanced or produces from the colibacillary possible glutamate receptor (particularly intestinal bacteria ybhL protein superfamily) or the activity of its homologue.

The sequence of e. coli k12 b4063 has been published in Blattner etc., and Science 277 (5331), 1453-1474, and 1997, and its activity is defined as the transcriptional activation agent of super-oxide reaction.Therefore, in one embodiment, the inventive method comprises as shown here transcribes protein, the transcriptional activation agent of particularly super-oxide reaction or the purposes of its homologue that control, DNA are regulated in conjunction with, inorganic chemical reagent opposing (for example heavy metal), amino acid metabolism adjusting and/or nitrogen and sulphur utilization from colibacillary participation, and it is used for producing each fine chemicals in biological or its part and is vitamin-E, particularly increases alpha-tocopherol, the preferred quantity of free or combining form alpha-tocopherol as described.In one embodiment, in the methods of the invention, the activity of the transcriptional activation agent (particularly intestinal bacteria ybhL protein superfamily) of described activity such as super-oxide reaction is enhanced or produces, and for example the transcriptional activation agent (particularly intestinal bacteria ybhL protein superfamily) or the activity of its homologue from colibacillary super-oxide reaction is enhanced or produces.

The sequence of e. coli k12 b0112 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be aromatic amino acid translocator (APC family).Therefore, in one embodiment, the inventive method comprise as shown here from colibacillaryly have amino acid transport body activity, the active protein of cell traffic, preferably from arginine permease super tame protein, particularly aromatic amino acid translocator (APC family) or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part and is vitamin-E, particularly increases alpha-tocopherol, the preferred quantity of free or combining form alpha-tocopherol as described.In one embodiment, in the methods of the invention, described activity such as APC family protein, particularly aromatic amino acid translocator, the particularly activity of arginine permease superfamily are enhanced or produce, and for example the activity from colibacillary APC family protein, particularly aromatic amino acid translocator, particularly arginine permease superfamily or its homologue is enhanced or produces.

The sequence of e. coli k12 b0986 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be lipoprotein.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary lipoprotein, preferably from the lipoprotein of putative protein matter b1706 superfamily or the purposes of its homologue, and it is used for producing each fine chemicals in biological or its part and is vitamin-E, particularly increases 5,8-dimethyl tocol and/or Gamma-Tocopherol, the preferred quantity of free or combining form 5,8-dimethyl tocol and/or Gamma-Tocopherol as described.In one embodiment, in the methods of the invention, described activity such as lipoprotein, the particularly activity of the lipoprotein of putative protein matter b1706 superfamily are enhanced or produce, and for example are enhanced or produce from the lipoprotein of colibacillary lipoprotein, particularly putative protein matter b1706 superfamily or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YFL019C has been published in Murakami Y. etc., Nat.Genet.10:261-268 (1995), and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes with the active protein of YFL019C or its homologue as shown here, it is used for by increasing vitamin-E precursor 2 as described, and the biosynthesizing of 3-dimethyl-5-phytyl chinol produces each fine chemicals in biological or its part be vitamin-E, preferred free or combining form vitamin-E.In a further embodiment, another gene of YFL019C protein expression and vitamin-E biosynthetic pathway, preferably participate in from middle product 2 with coding, the proteinic gene that 3-dimethyl-5-phytyl chinol produces vitamin-E is enhanced together.In one embodiment, in the methods of the invention, the activity of described YFL019C is enhanced or produces, and for example is enhanced or produces from the YFL019C of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YFL013C has been published in Murakami, Y etc., Nat.Genet.10 (3), 261-268 (1995) and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity is defined as INO80 chromatin reconstitution complex subunit.Therefore, in one embodiment, the inventive method comprises as shown here from the active protein of the colibacillary INO80 of having chromatin reconstitution complex subunit, particularly the INO80 chromatin reconstitution complex subunit of possible membranin YFL013c superfamily or the purposes of its homologue, it for example is used for by increasing vitamin-E precursor 2 as described, and 3-dimethyl-5-phytyl chinol biosynthesizing produces each fine chemicals in biological or its part be vitamin-E, preferred free or combining form vitamin-E.In a further embodiment, another gene of YFL013C protein expression and vitamin-E biosynthetic pathway, preferably participate in from middle product 2 with coding, the proteinic gene that 3-dimethyl-5-phytyl chinol produces vitamin-E is enhanced together.In one embodiment, in the methods of the invention, the activity of described activity such as INO80 chromatin reconstitution complex subunit is enhanced or produces, and for example is enhanced or produces from the INO80 chromatin reconstitution complex subunit of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YPL268W has been published in Bussey, H. etc., Nature 387 (6632 supplementary issue), 103-105 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be the phosphoinositide Phospholipid hydrolase.Therefore, in one embodiment, the inventive method comprises participation lipid as shown here, lipid acid and isoprenoid decompose, cell growth and form take place, the mitotic cell cycle, cell cycle control, the protein that intracellular signal transduction activates or DNA processes, yeast 1-phosphatidylinositols-4 particularly, the purposes of 5-bisphosphate phosphodiesterase superfamily (preferably having the phosphoinositide phospholipase activity) or its homologue, it is used for for example by increasing vitamin-E precursor 2 as described, 3-dimethyl-5-phytyl chinol biosynthesizing produces each fine chemicals in biological or its part be vitamin-E, preferably free or combining form vitamin-E.In a further embodiment, another gene of YPL268W protein expression and vitamin-E biosynthetic pathway, preferably participate in from middle product 2 with coding, the proteinic gene that 3-dimethyl-5-phytyl chinol produces vitamin-E is enhanced together.In one embodiment, in the methods of the invention, described activity such as yeast 1-phosphatidylinositols-4,5-bisphosphate phosphodiesterase superfamily protein or preferably have the yeast 1-phosphatidylinositols-4 of phosphoinositide phospholipase activity, 5-bisphosphate phosphodiesterase superfamily activity of proteins is enhanced or produces, for example from the 1-phosphatidylinositols-4 of yeast saccharomyces cerevisiae, the activity of 5-bisphosphate phosphodiesterase superfamily protein or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YFL053W has been published in Murakami, Y etc., Nat.Genet.10 (3), 261-268 (1995) and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be dihydroxyacetone kinase.Therefore, in one embodiment, protein, particularly the lyerone kinases DAK1 superfamily that the inventive method comprises participation c-compound as shown here and carbohydrate utilization, coerces reaction, modify by phosphorylation or dephosphorylation protein (preferably having the dihydroxyacetone kinase activity) or the purposes of its homologue, as described its to be used for producing each fine chemicals in biological or its part be vitamin-E, for example Gamma-Tocopherol and/or 5,8-dimethyl tocol, preferably free or combining form Gamma-Tocopherol and/or 5,8-dimethyl tocol.In one embodiment, in the methods of the invention, being enhanced or producing of described activity such as glyerone kinases DAK1 superfamily protein (preferably having the dihydroxyacetone kinase activity) for example is enhanced or produces from the glyerone kinases DAK1 superfamily protein (preferably having the dihydroxyacetone kinase activity) of yeast saccharomyces cerevisiae or the activity of its homologue.

The homologous compound (=homologue) of [0023.0.9.9] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.

Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with at least a Table II the 3rd row, 89-102 is capable and/or 472-482 capable shown in protein, for example have by contain Table I the 5th or 7 row, 89-102 is capable and/or 472-482 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, Table II the 3rd row, be respectively shown in the 89th or 90 or 482 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred vitamin E content increases.In one embodiment, homologue is the homologue that has sequence shown in Table I or II the 7th row, the 89th or 90 or 482 row respectively.In one embodiment, Table II the 3rd row, be respectively shown in the 89th or 90 or 482 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 89th or 90 or 482 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 89th or 90 or 482 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 89th or 90 or 482 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 89th or 90 or 482 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 89th or 90 or 482 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 89th or 90 or 482 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II the 3rd row, the 96th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred vitamin E, preferred 5,8-dimethyl tocol content increase.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 96th row.In one embodiment, shown in Table II the 3rd row, the 96th row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 96th row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 96th row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 96th row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 96th row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 96th row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 96th row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II the 3rd row, the 101st row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred vitamin E, preferred Gamma-Tocopherol content increase.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 101st row.In one embodiment, shown in Table II the 3rd row, the 101st row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 101st row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 101st row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 101st row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 101st row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 101st row is to have same or similar activity and from the Saccharomycetaceae homologue.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 101st row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II the 3rd row, the 91st or 92 row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, the increase of preferred vitamin E (by its precursor 2,3-dimethyl-5-phytyl chinol) content.In one embodiment, homologue is the homologue that has Table I or II the 7th row, is respectively sequence shown in the 91st or 92 row.In one embodiment, shown in Table II the 3rd row, the 91st or 92 row in the polypeptide homologue of any one from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 91st or 92 row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 91st or 92 row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 91st and 92 row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 91st or 92 row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 91st or 92 row is to have same or similar activity and from the homologue of Escherichia.

In one embodiment, shown in Table II the 3rd row, the 93rd, 94,95,472,473,474 or 475 row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred alpha-tocopherol content increase.In one embodiment, homologue is the homologue that has Table I or II the 7th row, is respectively sequence shown in the 93rd, 94,95,472,473,474 or 475 row.In one embodiment, shown in Table II the 3rd row, the 93rd, 94,95,472,473,474 or 475 row in the polypeptide homologue of any one from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 93rd, 94,95,472,473,474 or 475 row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 93rd, 94,95,472,473,474 or 475 row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 93rd, 94,95,472,473,474 or 475 row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 93rd, 94,95,472,473,474 or 475 row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 93rd, 94,95,472,473,474 or 475 row is to have same or similar activity and from the homologue of Escherichia.

In one embodiment, shown in Table II the 3rd row, 97-100 or 476 or 477 row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred 5,8-dimethyl tocol content increase.In one embodiment, homologue is the homologue that has Table I or II the 7th row, is respectively sequence shown in 97-100 or 476 or 477 row.In one embodiment, shown in Table II the 3rd row, 97-100 or 476 or 477 row in the polypeptide homologue of any one from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 97-100 or 476 or 477 row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 97-100 or 476 or 477 row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 97-100 or 476 or 477 row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 97-100 or 476 or 477 row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 97-100 or 476 or 477 row is to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, the 102nd row or 478-481 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred Gamma-Tocopherol content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively the 102nd row or 478-481 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, the 102nd row or 478-481 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, the 102nd row or 478-481 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, the 102nd row or 478-481 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, the 102nd row or 478-481 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, the 102nd row or 478-481 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, the 102nd row or 478-481 capable shown in the homologue of polypeptide be to have the homologue of same or similar activity from Escherichia.

[0023.1.9.9] as the homologue of the polypeptide shown in Table II the 3rd row, 89-102 or 472-482 are capable can be by Table I the 7th row, 89-102 or 472-482 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 89-102 or 472-482 capable shown in polypeptide.As the homologue of polypeptide shown in Table II the 3rd row, 89-102 or 472-482 are capable can be by Table I the 7th row, 89-102 or 472-482 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 89-102 or 472-482 capable shown in polypeptide.

The homologue of polypeptide can be to have to increase by 2 shown in Table II the 3rd row, 89-92 or 482 row, 3-dimethyl-5-phytyl chinol content and/or quantity is active, by polypeptide shown in Table I the 7th row, the polypeptide that is respectively nucleic acid molecule encoding shown in 89-92 or 482 row or Table II the 7th row, 89-92 or 482 row.The homologue of polypeptide can be to have to increase by 2 shown in Table II the 3rd row, 89-92 or 482 row, 3-dimethyl-5-phytyl chinol content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, 89-92 or 482 row or Table II the 7th row, 89-92 or 482 row.

The homologue of polypeptide shown in Table II the 3rd row, 93-95 or 472-475 are capable can be can be have increase alpha-tocopherol content and/or quantity is active, by Table I the 7th row, be respectively 93-95 or 472-475 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 93-95 or 472-475 capable shown in polypeptide.The homologue of polypeptide shown in Table II the 3rd row, 93-95 or 472-475 are capable can be have increase alpha-tocopherol content and/or quantity is active, by Table I the 7th row, 93-95 or 472-475 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 93-95 or 472-475 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, 96-100 or 476-477 are capable can be have increase 5,8-dimethyl tocol content and/or quantity is active, by Table I the 7th row, be respectively 96-100 or 476-477 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 96-100 or 476-477 capable shown in polypeptide.The homologue of polypeptide shown in Table II the 3rd row, 96-100 or 476-477 are capable can be have increase 5,8-dimethyl tocol content and/or quantity is active, by Table I the 7th row, 96-100 or 476-477 capable shown in the polypeptide of nucleic acid molecule encoding or table u the 7th row, 96-100 or 476-477 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, 101-102 or 478-481 are capable can be have increase Gamma-Tocopherol content and/or quantity is active, by Table I the 7th row, be respectively 101-102 or 478-481 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 101-102 or 478-481 capable shown in polypeptide.The homologue of polypeptide shown in Table II the 3rd row, 101-102 or 478-481 are capable can be have increase Gamma-Tocopherol content and/or quantity is active, by Table I the 7th row, 101-102 or 478-481 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 101-102 or 478-481 capable shown in polypeptide.

[0024.0.0.9] sees [0024.0.0.0]

[0025.0.9.9] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause biology or its part, vitamin-E or its precursor 2 in the preferred described biomass cells, 3-dimethyl-5-phytyl chinol level increases, alpha-tocopherol particularly, 5,8-dimethyl tocol and/or Gamma-Tocopherol level increase respectively, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in any one proteinic biology or the enzyme activity, if promptly with Table II the 3rd row, the 89th, 90,96, any one yeast saccharomyces cerevisiae protein shown in 101 or 482 row is compared and/or is listed as with Table II the 3rd, 91-95,97-100,102 or 472-481 capable shown in any one e. coli k12 protein compare, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as each fine chemicals of increase in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

[0025.1.0.9] sees [0025.1.0.0]

[0025.2.0.9] sees [0025.2.0.0]

[0025.3.18.18] in one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and is expressed in evolution with in the eozoan biology far away, the used polypeptide of polypeptide of the present invention or the inventive method is still given described activity, as increase each fine chemicals in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

[0026.0.0.9] to [0033.0.0.9]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.9.9] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, it has Table II the 3rd row, be respectively 89-92 or 482 the row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or the 101st the row and/or 102 and/or 478-481 capable shown in protein, or by Table I the 5th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or the 101st row and/or 102 and/or 478-481 capable shown in the protein of nucleic acid molecule encoding, or its homologue is (as Table I the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in homologue) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows each fine chemicals quantity that increases.

[0035.0.0.9] to [0044.0.0.9]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.9.9] is at e. coli k12 protein b0161 or its homologue (shown in Table I the 5th or 7 row, the 472nd row), for example have under the situation that the active activity of proteins that defines is enhanced in [0022.0.9.9], preferably, give in one embodiment fine chemicals, preferred alpha-tocopherol be increased in 50% and 138% or more between.

At e. coli k12 protein b0970 or its homologue (shown in Table I the 5th or 7 row, the 473rd row), for example have under the situation that the active activity of proteins that defines is enhanced in [0022.0.9.9], preferably, give in one embodiment fine chemicals, preferred alpha-tocopherol be increased in 47% and 54% or more between.

At e. coli k12 protein b4063 or its homologue (shown in Table I the 5th or 7 row, the 475th row), for example have under the situation that the active activity of proteins that defines is enhanced in [0022.0.9.9], preferably, give in one embodiment fine chemicals, preferred alpha-tocopherol be increased in 16% and 47% or more between.

At e. coli k12 protein b0785 or its homologue (shown in Table I the 5th or 7 row, the 476th or 479 row), for example have under the situation that the active activity of proteins that defines is enhanced in [0022.0.9.9], preferably, give in one embodiment fine chemicals, preferred β-and/or Gamma-Tocopherol be increased in 34% and 40% or more between.

At e. coli k12 protein b3938 or its homologue (shown in Table I the 5th or 7 row, the 477th or 481 row), for example have under the situation that the active activity of proteins that defines is enhanced in [0022.0.9.9], preferably, give in one embodiment fine chemicals, preferred β-and/or Gamma-Tocopherol be increased in 50% and 120% or more between.

In one embodiment, e. coli k12 protein b3160 (as Table I the 5th or 7 row, the 474th the row shown in), the active protein or its homologue that perhaps have the monooxygenase with luciferase sample atpase activity of the supposition of being defined as, for example under the situation that the activity of transcriptional is enhanced, preferably, giving the fine chemicals alpha-tocopherol in one embodiment is increased between 47% and 91%.

In one embodiment, at e. coli k12 protein b1829 or its homologue, for example have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 91st row) of heat shock protein or protease activity, give each fine chemicals, preferred vitamin E precursor 2,3-dimethyl-5-phytyl chinol be increased in 48% and 283% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, for example have under the activity situation about being enhanced of heat shock protein (for example shown in Table I the 5th or 7 row, the 94th row) of protease activity, give each fine chemicals, preferred vitamin E, particularly alpha-tocopherol be increased in 39% and 90% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, for example have under the activity situation about being enhanced of heat shock protein (for example shown in Table I the 5th or 7 row, the 100th or 102 row) of protease activity, give each fine chemicals, preferred vitamin E, particularly 5,8-dimethyl tocol and/or Gamma-Tocopherol be increased in 40% and 325% or more between.

In one embodiment, at e. coli k12 protein b1827 or its homologue, what for example have supposition has under the situation that DNA is enhanced in conjunction with the activity of the active protein of the transcription repressor (IcIR family) in wing spirane structure territory (for example shown in Table I the 5th or 7 row, the 92nd row), give each fine chemicals, preferred vitamin E precursor 2,3-dimethyl-5-phytyl chinol be increased in 76% and 91% or more between.

In one embodiment, at e. coli k12 protein b1827 or its homologue, what for example have supposition has under the situation that DNA is enhanced in conjunction with the activity of the active protein of the transcription repressor (IcIR family) in wing spirane structure territory (for example shown in Table I the 5th or 7 row, the 97th row), give each fine chemicals, preferred vitamin E, particularly 5,8-dimethyl tocol be increased in 34% and 116% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example have the DNA chain exchange that has proteolytic enzyme and nuclease and recombinant protein active protein (for example as Table I the 5th or 7 row, the 93rd capable shown in) activity situation about being enhanced under, give each fine chemicals, preferred vitamin E, particularly alpha-tocopherol be increased in 51% and 196% or more between.

In one embodiment, at e. coli k12 protein b0112 or its homologue, for example have under the activity situation about being enhanced of the active protein of aromatic amino acid translocator (APC family) (for example shown in Table I the 5th or 7 row, the 95th row), give each fine chemicals, preferred vitamin E, particularly alpha-tocopherol be increased in 43% and 63% or more between.

In one embodiment, at e. coli k12 protein b0175 or its homologue, for example under the activity situation about being enhanced of CDP-diester glycerine synthase (for example shown in Table I the 5th or 7 row, the 99th or 478 row), give each fine chemicals, preferred vitamin E, particularly 5,8-dimethyl tocol and/or Gamma-Tocopherol be increased in 34% and 40% or more between.

In one embodiment, at e. coli k12 protein b0986 or its homologue, for example under the activity situation about being enhanced of lipoprotein (for example shown in Table I the 5th or 7 row, the 98th and 480 row), give each fine chemicals, preferred vitamin E, particularly 5,8-dimethyl tocol and/or Gamma-Tocopherol be increased in 35% and 61% or more between.

At yeast saccharomyces cerevisiae protein YFL019C or its homologue (shown in Table I the 5th or 7 row, the 482nd row), for example have under the situation that the active activity of proteins that defines is enhanced in [0022.0.9.9], preferably, give fine chemicals, preferred 2 in one embodiment, 3-dimethyl-5-phytyl chinol be increased in 97% and 440% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL013C or its homologue, for example under the activity situation about being enhanced of INO80 chromatin reconstitution complex subunit, particularly membranin YFL013c superfamily (for example shown in Table I the 5th or 7 row, the 90th row), give each fine chemicals, preferred vitamin E precursor 2,3-dimethyl-5-phytyl chinol be increased in 97% and 440% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPL268W or its homologue, for example phosphoinositide Phospholipid hydrolase, particularly yeast 1-phosphatidylinositols-4, under the activity situation about being enhanced of 5-bisphosphate phosphodiesterase superfamily (for example shown in Table I the 5th or 7 row, the 89th row), give each fine chemicals, preferred vitamin E precursor 2,3-dimethyl-5-phytyl chinol be increased in 44% and 51% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL053W or its homologue, for example under the activity situation about being enhanced of dihydroxyacetone kinase, particularly glyerone kinases DAK1 superfamily (for example shown in Table I the 5th or 7 row, the 96th and 101 row), give each fine chemicals, preferred vitamin E, particularly Gamma-Tocopherol and/or 5,8-dimethyl tocol be increased in 45% and 123% or more between.

[0046.0.9.9] in one embodiment, as disclosed e. coli k12 protein b0161 or its homologue in [0016.0.9.9] (as Table I the 5th or 7 row, the 472nd the row shown in), for example have such as in [0022.0.9.9] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferred alpha-tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, as disclosed e. coli k12 protein b0970 or its homologue in [0016.0.9.9] (as Table I the 5th or 7 row, the 473rd the row shown in), for example have such as in [0022.0.9.9] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferred alpha-tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, as disclosed e. coli k12 protein b3160 or its homologue in [0016.0.9.9] (as Table I the 5th or 7 row, the 474th the row shown in), for example have such as in [0022.0.9.9] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferred alpha-tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, as disclosed e. coli k12 protein b4063 or its homologue in [0016.0.9.9] (as Table I the 5th or 7 row, the 475th the row shown in), for example have such as in [0022.0.9.9] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferred alpha-tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, as disclosed e. coli k12 protein b0785 or its homologue in [0016.0.9.9] (as Table I the 5th or 7 row, the 476th and 479 the row shown in), for example have such as in [0022.0.9.9] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferred 5,8-dimethyl tocol and/or Gamma-Tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, as disclosed e. coli k12 protein b3938 or its homologue in [0016.0.9.9] (as Table I the 5th or 7 row, the 477th and 481 the row shown in), for example have such as in [0022.0.9.9] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferred 5,8-dimethyl tocol and/or Gamma-Tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at e. coli k12 protein b1829 or its homologue, for example have under the activity situation about being enhanced of heat shock protein (for example shown in Table I the 5th or 7 row, the 91st, 94 or 100 row) of protease activity, give each fine chemicals, preferred alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol and/or 2,3-dimethyl-5-phytyl chinol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at e. coli k12 protein b1827 or its homologue, what for example have supposition has under the situation about being enhanced of DNA in conjunction with the active protein of the transcription repressor (IcIR family) in wing spirane structure territory (for example shown in Table I the 5th or 7 row, the 92nd or 97 row), preferably, give each fine chemicals, preferred 5,8-dimethyl tocol and/or 2,3-dimethyl-5-phytyl chinol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example have the DNA chain exchange that has proteolytic enzyme and nuclease and recombinant protein active protein (for example as Table I the 5th or 7 row, the 93rd capable shown in) activity situation about being enhanced under, preferably, give each fine chemicals, preferred alpha-tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at e. coli k12 protein b0112 or its homologue, for example have under the activity situation about being enhanced of the active protein of aromatic amino acid translocator (APC family) (for example shown in Table I the 5th or 7 row, the 95th row), preferably, give each fine chemicals, preferred alpha-tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at e. coli k12 protein b0175 or its homologue, for example under the activity situation about being enhanced of CDP-diester glycerine synthase (for example shown in Table I the 5th or 7 row, the 99th or 478 row), preferably, give each fine chemicals, preferred 5,8-dimethyl tocol and/or Gamma-Tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at e. coli k12 protein b0986 or its homologue, for example under the activity situation about being enhanced of lipoprotein (for example shown in Table I the 5th or 7 row, the 98th and 480 row), preferably, give each fine chemicals, preferred 5,8-dimethyl tocol and/or Gamma-Tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL013C or its homologue, for example under the activity situation about being enhanced of INO80 chromatin reconstitution complex subunit, particularly membranin YFL013c superfamily (for example shown in Table I the 5th or 7 row, the 90th row), preferably, give each fine chemicals, preferred 2,3-dimethyl-5-phytyl chinol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL053W or its homologue, for example under the activity situation about being enhanced of dihydroxyacetone kinase, particularly glyerone kinases DAK1 superfamily (for example shown in Table I the 5th or 7 row, the 96th or 101 row), preferably, give each fine chemicals, preferred β-and/or Gamma-Tocopherol and have active other compound of vitamin-E or its precursor increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YPL268W or its homologue, for example phosphoinositide Phospholipid hydrolase, particularly yeast 1-phosphatidylinositols-4, under the activity situation about being enhanced of 5-bisphosphate phosphodiesterase superfamily (for example shown in Table I the 5th or 7 row, the 89th row), preferably, give each fine chemicals, preferred 2,3-dimethyl-5-phytyl chinol and have active other compound of vitamin-E or its precursor increases.

[0047.0.0.9] to [0048.0.0.9]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.9.9] has raising of giving 2, and 3-dimethyl-5-phytyl chinol quantity or the active protein of level preferably have the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in 89-92 or 482 row and/or as Table II the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in 89-92 or 482 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in 89-92 or 482 row) sequence of coded polypeptide, and have and give β-2, the activity that 3-dimethyl-5-phytyl chinol level increases described in the literary composition.

Have to give and improve alpha-tocopherol quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in 93-95 or 472-475 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 93-95 or 472-475 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 93-95 or 472-475 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the alpha-tocopherol level of giving described in the literary composition increases.

Have to give and improve 5,8-dimethyl tocol quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in 96-100 or 476-477 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 96-100 or 476-477 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 96-100 or 476-477 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the 5,8-dimethyl tocol level of giving described in the literary composition increases.

Have to give and improve Gamma-Tocopherol quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in 101-102 or 478-481 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 101-102 or 478-481 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 101-102 or 478-481 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the Gamma-Tocopherol level of giving described in the literary composition increases.

[0050.0.9.9] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol sylvite or sodium salt or its ester separately.

[0051.0.9.9] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise vitamin-E or its precursor 2, the composition of 3-dimethyl-5-phytyl chinol.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce multivitamin E compound or its precursor 2, the composition or the mixture of 3-dimethyl-5-phytyl chinol.

[0052.0.0.9] sees [0052.0.0.0]

[0053.0.9.9] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 is capable or 478-481 capable shown in) active polypeptide) express to increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA give nucleic acid molecule of the present invention or the coded protein of the inventive method nucleic acid molecule used therefor (for example has as Table II the 3rd row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein is given has the active of each fine chemicals of the raising described in the literary composition, (for example have by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide as Table II the 3rd row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by nucleic acid molecule of the present invention or the coded protein of the inventive method nucleic acid molecule used therefor, polypeptide perhaps of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in) active polypeptide) express and improve;

(e) by add the activity that one or more external source inducible factors come stimulating protein in biological or its part, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in) active polypeptide) express and improve;

(f) transgenosis of expression coded protein, described protein is given the activity with each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, and described nucleic acid molecule encoding has the active of each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method as Table II the 3rd row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in) active polypeptide);

(h) the negative Expression element of positive Expression element or removal improves code book invention polypeptide or the used polypeptide of the inventive method (for example has as Table II the 3rd row by adding, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.9.9] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having as Table II the 5th row, be respectively 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in protein or its homologue (Table II the 7th row for example, be respectively 89-92 or 482 row or 93-95 or 472-475 is capable 96-100 or 476-477 is capable or 101-102 or 478-481 capable shown in polypeptide) give each fine chemicals after active expression of polypeptides or the activity and increase.

[0055.0.0.9] to [0067.0.0.9]: see that [0055.0.0.0] is to [0067.0.0.0]

With can not be to vitamin-E or its precursor 2, the generation of 3-dimethyl-5-phytyl chinol causes the mode of disadvantageous effect to introduce sudden change [0068.0.9.9].

[0069.0.0.9] sees [0069.0.0.0]

[0070.0.9.9] will be owing to will give nucleic acid molecule of the present invention or a gene or a plurality of gene (nucleic acid construct of mentioning for example) of polypeptide of the present invention or the expression of the inventive method nucleic acid molecule used therefor, perhaps a gene of code book invention protein or the used polypeptide of the inventive method or a plurality of gene import biology separately or with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, the vitamin-E or its precursor 2 that for example contain high level (from the physiology of nutrition angle), the favourable vitamin composition of 3-dimethyl-5-phytyl chinol.

[0071.0.0.9] sees [0071.0.0.0]

[0072.0.9.9] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has other VITAMIN or provitamin or carotenoid except vitamin-E or its precursor 2 outside 3-dimethyl-5-phytyl chinol.

[0073.0.9.9] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or the used polypeptide of the inventive method or its homologue (for example Table II the 5th or 7 row, 89-92 or 482 row or 93-95 472-475 is capable or 96-100 476-477 is capable or 101-102 or 478-481 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and optional other free and/or bonded vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol.

[0074.0.9.9] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded or free and each fine chemicals of bonded, if and dissociate or/and bonded vitamin-E or its precursor 2 3-dimethyl-5-phytyl chinol as selecting to produce, reclaim and expect to separate other.

[0075.0.0.9] to [0077.0.0.9]: see that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.9.9] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes, for example according to 6,399,059 (Thermally stable enzyme composition and methodof preparing the same); 6,361,800 (Multi-vitamin and mineral supplement, 6,348,200:Cosmetic composition); 6,338,854 (Photoaging skin-carepreparation and method for the treatment of wrinkled skin); 6,323,188 (Treatmentand prevention of cardiovascular diseases, heart attack, and stroke, primary and subsequent, with help of aspirin and certain vitamins); 6,299,896 (Multi-vitamin and mineral supplement); 6,262,279 (Preparationof tocopherols); 6,362,221 (Compositions containing natural Lyeopene andnatural tocopherol); 6,358,997 (Tocopherol and tocotrienolcompositions); 6,344,573 (Process for extraction and concentration ofliposoluble vitamins and provitamins, growth factors and animal andvegetable hormones from residues and by-products of industrializedanimal and vegetable products); 6,242,227 (Method of vitaminproduction); 6,207,187 (Compositions based on tocopherols); Disclosing among 6,177,114 (the Refining of edible oil rich in natural carotenes and Vitamin Ewhich are expressly in corporated herein by reference).Can or pass through those skilled in the art method purified fermentation broth, tunning, plant or plant product known and that describe hereinafter as above-mentioned application.

At generation vitamin-E according to the present invention or its precursor 2, in the method for 3-dimethyl-5-phytyl chinol, preferably to gathering in the crops described biology after the culturing step of genetic modified organism (hereinafter being also referred to as genetically modified organism) and from described bioseparation vitamin-E.

Known and be applicable to that the mode of particular organisms gathers in the crops biology with itself.By the microorganism of fermentation culture, for example bacterium, mosses, yeast and fungi or vegetable cell can reclaim in the liquid medium within, for example by centrifugal, decant or filtered and recycled.Plant grown on the solid medium in a manner known way and results correspondingly.

In a manner known way, for example by extracting and (in due course) other chemistry or physical purification process, for example intermediate processing, crystallography, thermal separation method (as rectificating method) or physical separation method (for example chromatography) from results biomass separating VE or its precursor 2,3-dimethyl-5-phytyl chinol.

Separating VE from oil plant for example preferably separates from vegetables oil or from the vapor distillation thing (deodorizing condensation product) that obtains the vegetables oil deodorizing with distillation by chemical transformation.

The other method of separating VE is described in for example DE 31 26 110 A1, EP 171 009 A2, GB 2 145 079, EP 333 472 A2 and WO 94/05650 from the deodorizing condensation product.

For these different methods of setting up gradually, product is vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, for example comprise alpha-tocopherol, 5,8-dimethyl tocol or Gamma-Tocopherol or vitamin-E or its precursor 2, the composition of 3-dimethyl-5-phytyl chinol, the composition that for example contains alpha-tocopherol, 5,8-dimethyl tocol or Gamma-Tocopherol, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.9] to [0084.0.0.9]: see that [0079.0.0.0] is to [0084.0.0.0]

[0084.1.9.9] the present invention has also considered such embodiment, i.e. vitamin-E or its precursor 2 in the generation of each fine chemicals, 3-dimethyl-5-phytyl chinol or other vitamin-E precursor compound are present in selected phytosynthesis active bio as the host; Even if for example cyanobacteria, liver moss, algae or also can produce the plant of vitamin-E for wild-type.The present invention has also considered such embodiment, i.e. be deficient in vitamin E or its precursor 2 of host, 3-dimethyl-5-phytyl chinol or other vitamin-E or its precursor 2, the precursor of 3-dimethyl-5-phytyl chinol, for example Vinca (Vinca).In the plant of latter's type, the DNA that is inserted comprises that coding produces the protein of vitamin-E precursor (can biologically be transformed into the compound with the active compound of vitamin-E) and the gene of one or more modifying enzyme (not existing at first) in this plant.

Preferred monocotyledons is selected from the unifacial leaf crop plants especially, and Gramineae for example is as rice, corn, wheat or other cereal such as barley, millet, rye, triticale or oat and sugarcane and all careless classes.The present invention is specially adapted to the dicotyledons biology.Preferred dicotyledons is selected from dicotyledonous crop plants especially, for example

-composite family (Asteraceae), for example Sunflower Receptacle, Tagetes or calendulin and many other genus,

-composite family (Compositae), particularly Lactuca, lettuce and many other kinds very in particular,

-Cruciferae (Cruciferae), particularly Btassica, colea (rape), rape (Brassica campestris) (beet), wild cabbage (for example Caulis et Folium Brassicae capitatae (cabbage), Cauliflower (cauliflower) or cabbage (broccoli) and other Caulis et Folium Brassicae capitatae type) very in particular; And mouse ear mustard belongs to, very particularly Arabidopis thaliana and Chinese celery (cress) or rape (canola) and many other species,

-Curcurbitaceae, for example muskmelon (melon), pumpkin, zucchini (zucchini) and many other species,

-pulse family, particularly Glycine, soybean, soja and alfalfa, pea, beans or peanut and many other species very in particular,

-Rubiaceae, preferred lip subclass (Lamiidae), for example fruitlet coffee or fruit coffee (coffee plants) and many other species greatly,

-Solanaceae, particularly tomato belong to, tomato (tomato) and Solanum very in particular, and potato and eggplant (eggplant (aubergine)) and Capsicum very in particular, capsicum (red pepper) and tobacco and many other species very in particular,

-Sterculiaceae, preferred Dilleniidae (Dilleniidae), for example cocoa tree (cocoa plants) and many other species,

-Theaceae, preferred Dilleniidae, for example tea (Camellia sinensis) or tea (Theasinensis) (tea tree plant) and many other species,

-umbelliferae (Umbelliferae), particularly Daucus, Radix Dauci Sativae and apium (Apium), celery (Apium graveolens var.dulce) (celeriac dish (celeriac)) and many other species very in particular very in particular;

And linseed oil, soybean, cotton, hemp, flax, cucumber, spinach, Radix Dauci Sativae, sugar beet and multiple tree, nut and grape, particularly banana and Kiwifruit.

Also consider decorative plant, prolification tree or decorated tree, flower, cut-flower, shrub or turf.The non-limiting example that can mention is angiosperm, bryophyte, for example Hepaticae (Hepaticae) (liverwort (liverworts)) and moss guiding principle (Musci) (moss (mosses)); Pteridophyte is as fern, scouring rush and lycopod; Gymnosperm is as conifer, cycad, ginkgo and Gnetales (gnetales), the Rosaceae, as rose, Ericaceae is as rhododendron and rhododendron, Euphorbiaceae is as poinsettia and crotons, Caryophyllaceae, as carnation, Solanaceae is as petunia, Gesneriaceae (Gesneriaceae), as African violet, Balsaminaceae (Balsaminaceae), as Flower of Garden Balsam, the orchid family is as orchid, Iridaceae is as gladiolus, iris, xiangxuelan and Stigma Croci, composite family, as mary bush, Mang ox seedling section is as the blue certain herbaceous plants with big flowers in sky, Liliaceae, as dragon tree, Moraceae (Moraceae), as Ficus, Araeceae (Araceae) is as Philodendron (philodendron) and other.

Particularly preferred plant biological is to be selected from the Borrago officinalis, rape, colea, overgrown with weeds blue or green, hemp (Cannabis sativa), safflower, coconut, Crambe (Crambe abyssinica), calyx is apart from flower (Cuphea species), oil palm, Ekeis oleiferu, soybean, upland cotton, sea island cotton, cotton, Sunflower Receptacle (Helianthus annus), flax, root of Redsepal Eveningprimrose (Oenothera biennis), Ozeaeuropea, rice (Oryza sativa), castor-oil plant, flax, wheat (Triticum species), Zea mays, the oil-produced vegetable of English walnut and apricot.

In one embodiment, preferred plant includes but not limited to Flower of Aztec Marigold, colea, tobacco (Nicotinana tabacum), Sunflower Receptacle, rape, Solanum (potato) and soybean.

Preferred cyanobacteria is synechocystis (Synechocystis) cyanobacteria, for example cytoalgae PCC6803 perhaps opens up leaf sword-like leave moss (Physcometrella patens) or some shape beads algae (Nostocpunctiforme) ATCC 29133, anabena (Anabaena sp.) PCC7120.

Preferred algae is a green alga, for example Chlorella (Chlorella), haematococcus (Haematococcus), Phaeodactylum tricornutum (Phaedactylum tricornatum), volvox (Volvox) or Dunaliella salina belong to (Dunaliella), particularly for example Spongiococcum sp. such as Spongiococcum exentricum, Chlorella certain (Chlorella sp.), haematococcus, Phaeodactylum tricornutum.

[0085.0.9.9] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.9] to [0087.0.0.9]: see that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.9.9] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Because the nutritive value that for example is used for the plant of raise poultry depends on said vitamin E and the total number of the vitamin-E of originating as feed, so this is very important for the plant breeder.In addition, because the antioxidant level of plant milk extract depends on said vitamin E and as the total number of the vitamin-E of antioxidant, so this production for make-up composition also is important.

[0088.1.0.9] sees [0088.1.0.0]

[0089.0.0.9] to [0090.0.0.9]: see that [0089.0.0.0] is to [0090.0.0.0]

[0091.0.9.9] therefore, the plant component foreign matter content is low as far as possible, and preferably other foreign matter contents are also low as far as possible, and the aforementioned vitamin-E that obtains or its precursor 2,3-dimethyl-5-phytyl chinol is pure form as far as possible.In these are used, the content of plant component favourable for by weight less than 10%, preferred 1%, more preferably 0.1%, very particularly preferably 0.01% or lower.

[0092.0.0.9] to [0094.0.0.9]: see that [0092.0.0.0] is to [0094.0.0.0]

[0095.0.9.9] advantageously increases described vitamin-E or its precursor 2 by method of the present invention in genetically modified organism, 3-dimethyl-5-phytyl chinol pond is to separate a large amount of pure basically fine chemicals.

[0096.0.9.9] is in another embodiment preferred of the present invention, improving nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as required fine chemicals such as vitamin-E in the biology or its precursor 2, the pond of 3-dimethyl-5-phytyl chinol) combines and can be used for producing each fine chemicals.

[0097.0.0.9] sees [0097.0.0.0]

[0098.0.9.9] in preferred embodiments, each fine chemicals (vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol) be produce according to the present invention and carry out isolating where necessary.The method according to this invention produces other VITAMIN, provitamin or carotenoid, for example carotene or xenthophylls, perhaps its mixture or with the mixture of other compound be favourable.

[0099.0.9.9] for microbial fermentation, aforementioned vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol can be accumulated in substratum and/or the cell.If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biological and substratum, separate and comprise each fine chemicals or vitamin-E or its precursor 2, the composition of 3-dimethyl-5-phytyl chinol.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.9.9] comprises the method according to this invention synthetic and comprises vitamin-E or its precursor 2, the transgenic plant of 3-dimethyl-5-phytyl chinol such as alpha-tocopherol, 5,8-dimethyl tocol or Gamma-Tocopherol can advantageously directly be put on market, and do not need institute's synthetic oils, lipid or lipid acid are separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

In plant, the biosynthesizing site of vitamin-E is leaf texture especially, and therefore separating leaf is meaning.Yet this is not restrictive, can also the tissue specificity mode take place in remaining plant part because express, particularly in the seed that comprises fat.Therefore, another embodiment preferred relates to vitamin-E or its precursor 2, and the seed-specific of 3-dimethyl-5-phytyl chinol separates.

Yet, each fine chemicals that the method according to this invention produces can also be with the form of its oils, fat, lipid as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free vitamin E or its precursor 2, and 3-dimethyl-5-phytyl chinol is from biology, advantageously separate from plant.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can by to plant part for example plant seed process or extract realization.In order to improve the efficient of extraction, with vegetable material, particularly seed clean, softening (temper) and to shell in case of necessity and peel off be favourable.For example, contain vitamin-E or its precursor 2, oils, fat and/or the lipid of 3-dimethyl-5-phytyl chinol can need not heat cold be beaten or cold-press process obtains by being called.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because vitamin-E or its precursor 2 in the microorganism, 3-dimethyl-5-phytyl chinol is positioned at cell, must relate to separation to biomass to their recovery.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.The mensuration of tocopherol was carried out description by Tan and Tsumura in the pair cell in 1989, be also shown in the Biotechnology of Vitamins that Erik J.Vandamme edits, Pigments and GrowthFactors, London, 1989, the 96-103 pages or leaves.Many other methods of measuring tocopherol content are well known by persons skilled in the art.

[0101.0.0.9] sees [0101.0.0.0]

[0102.0.9.9] for example, vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol can advantageously be analyzed and detect by MS or MSMS method by HPLC or GC separation method.Contain vitamin-E or its precursor 2 by using standard method of analysis GC, GC-MS or TLC can clearly detect to the biology analysis of recombinating, the existence of the product of 3-dimethyl-5-phytyl chinol, described analytical procedure is described (1997 by Christie and reference wherein, Advances onLipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gaschro matography/mass spectrometric method], Lipide 33:343-353).By ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling or by other applicable method with material fragmentation to be analyzed; Be also shown in the Biotechnology ofVitamins that Erik J.Vandamme edits, Pigments and Growth Factors, London, 1989, the 96-103 pages or leaves.

[0103.0.9.9] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as Table II the 5th or 7 row, are respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I the 5th or 7 row, is respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use have as Table III the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0103.1.9.9] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide of sequence.

[0103.2.9.9.] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one or more sequence differences be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one or more sequences form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIB the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide of sequence.

[0104.0.9.9] in one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention or the nucleic acid that is used for the inventive method not by Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide of sequence.

[0105.0.0.9] to [0107.0.0.9] sees that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.9.9] method of the present invention and had Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleic acid molecule of sequence, from Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in aminoacid sequence derive or from containing Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the polypeptide deutero-nucleic acid molecule of consensus sequence, perhaps its coding has as Table II the 3rd, 5 or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the enzymic activity of polypeptide or bioactive polypeptide or for example to give each fine chemicals behind its expression or active the increasing be vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, derivative or homologue that 5,8-dimethyl tocol and/or Gamma-Tocopherol increase.

[0109.0.0.9] sees [0109.0.0.0]

[0110.0.9.9] helps the inventive method and coding has the polypeptide of polypeptide of the present invention or the used polypeptide active of the used or of the present invention process of the inventive method (for example as Table II the 5th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in protein or by as Table I the 5th be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the protein of nucleic acid molecule encoding or its homologue (as Table I or II the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in)) nucleic acid molecule can from generally open database, determine.

[0111.0.0.9] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.9.9] the inventive method is the isolated nucleic acid sequences form, its coding has as Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide active polypeptide or have as Table II the 5th and 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the polypeptide of peptide sequence, and give vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, 5,8-dimethyl tocol and/or Gamma-Tocopherol increase.

[0113.0.0.9] to [0120.0.0.9]: see that [0113.0.0.0] is to [0120.0.0.0]

[0120.1.9.9] advantageously, the production bacterial strain of selecting in the inventive method is to be selected from following microorganism: green alga such as Spongioccoccum exentricum, Chlorella sorokiniana (Chlorella pyrenoidesa (pyrenoidosa) 7-11-05), perhaps haematococcus, Phaeodactylum tricornutum, the algae that volvox or Dunaliella salina belong to, perhaps fungi is as belonging to the fungi of colored otology (Daccrymycetaceae), perhaps non-photosynthetic bacterium such as methylotrophy bacterium, Flavobacterium, actinomycetes such as Streptomyceschrestomyceticus, mycobacterium such as Mycobacterium phlei (Mycobacterim phlei) or the red bacterium of pod membrane (Rhodobacter capsulatus).Therefore, the present invention has also considered such embodiment, be deficient in vitamin E or its precursor 2 of host wherein, 3-dimethyl-5-phytyl chinol or other vitamin-E or its precursor 2, the precursor of 3-dimethyl-5-phytyl chinol, for example Vinca.In the plant of latter's type, the DNA that is inserted comprises that coding produces the protein of vitamin-E precursor (can biologically be transformed into the compound with the active compound of vitamin-E) and the gene of one or more modifying enzyme (not existing at first) in this plant.

The present invention has also considered such embodiment, i.e. vitamin-E or its precursor 2 in the generation of each fine chemicals, 3-dimethyl-5-phytyl chinol or other vitamin-E precursor compound are present in selected phytosynthesis active bio as the host; Even if for example cyanobacteria, liver moss, algae or also can produce the plant of vitamin-E for wild-type.

[0121.0.9.9] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in peptide sequence or its functional homologue as herein described the difference of one or more amino acid moleculars is arranged, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, give 2 after the activity of peptide sequence shown in 89-92 or 482 row, 3-dimethyl-5-phytyl chinol level increases; Perhaps give the alpha-tocopherol level after the activity of peptide sequence shown in capable and increase improving Table II the 5th or 7 row, 93-95 or 472-475; Perhaps give the 5,8-dimethyl tocol level after the activity of peptide sequence shown in capable and increase improving Table II the 5th or 7 row, 96-100 or 476-477; Perhaps give the Gamma-Tocopherol level after the activity of peptide sequence shown in capable and increase improving Table II the 5th or 7 row, 101-102 or 478-481.

[0122.0.0.9] to [0127.0.0.9]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.9.9] polymerase chain reaction (PCR) amplification is used is (for example as Table III the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 primer shown in capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence or from as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence deutero-sequence produce.

[0129.0.9.9] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (sequence of polypeptide particularly of the present invention or the used polypeptide of the inventive method).The conservative region of polypeptide of the present invention or the used polypeptide of the inventive method is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the aminoacid sequence of a specific position in several homologues of different sources.Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in consensus sequence from described comparison.

[0130.0.9.9] can use degenerated primer to have the new protein fragment of aforementioned activity (give vitamin-E or its precursor 2 as comprise this segmental protein expression or activity in raising after, 3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol increase) by pcr amplification then.

[0131.0.0.9] to [0138.0.0.9]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.9.9] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna is under loose hybridization conditions and for 2, the Table I the 5th of 3-dimethyl-5-phytyl chinol or 7 row, 89-92 or 482 row, preferred Table I B the 7th row, sequence shown in 89-92 or 482 row or for the Table I the 5th of alpha-tocopherol or 7 row, 93-95 or 472-475 are capable, preferred Table I B the 7th row, sequence shown in 93-95 or 472-475 are capable or for the Table I the 5th of 5,8-dimethyl tocol or 7 row, 96-100 or 476-477 are capable, preferred Table I B the 7th row, sequence shown in 96-100 or 476-477 are capable or for the Table I the 5th of Gamma-Tocopherol or 7 row, 101-102 or 478-481 are capable, preferred Table I B the 7th row, sequence hybridization shown in 96-100 or 476-477 are capable, and the coding expression has each fine chemicals of increasing, be vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, the active peptide of 5,8-dimethyl tocol and/or Gamma-Tocopherol.

[0140.0.0.9] to [0146.0.0.9]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.9.9] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise one of the nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.9.9] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor contain following nucleotide sequence: with Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in nucleotide sequence or its portion homologous at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably has an aforementioned activity, particularly have after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue and increase vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, the activity of 5,8-dimethyl tocol and/or Gamma-Tocopherol.

[0149.0.9.9] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one of nucleotide sequence or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and (as gives vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, 5,8-dimethyl tocol and/or Gamma-Tocopherol increase) and the protein of protein active as shown in Table II randomly.

[00149.1.9.9] randomly, in one embodiment, with Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd be listed as, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 protein shown in capable is known activity or is used for these protein of note.

[0150.0.9.9] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly has aforementioned activity, for example when its active raising, give vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, 5,8-dimethyl tocol and/or Gamma-Tocopherol increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and described nucleotides sequence column region is listed as with Table I the 5th or 7 under stringent condition, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the sense strand of one of sequence, Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotide hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the right PCR of primer will produce as Table I the 5th or 7 and be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in fragment or its gene product of polynucleotide sequence.

[0151.0.0.9] sees [0151.0.0.0]

[0152.0.9.9] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coded polypeptide or its part, described polypeptide or its part comprise and are listed as Table II the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the abundant homology of aminoacid sequence, make this albumen or its part keep the ability that participates in producing each fine chemicals, specifically be included in plant or the microorganism as described above or raising vitamin-E as be shown in the examples or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, the activity of 5,8-dimethyl tocol and/or Gamma-Tocopherol level.

[0153.0.9.9] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprises and is listed as Table II the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in protein or its part have Table II the 3rd row for example as herein described, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the activity of polypeptide.

[0154.0.9.9] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% also most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.9] to [0156.0.0.9]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.9.9] the present invention relates in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in consensus sequence polypeptide or as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 consensus sequence shown in capable or as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 consensus sequence shown in capable or as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet, in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, is respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in nucleic acid molecule identical.

[0158.0.0.9] to [0160.0.0.9]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.9.9] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.9] sees [0162.0.0.0]

[0163.0.9.9] preferably, under stringent condition with Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.9] sees [0164.0.0.0]

[0165.0.9.9] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I, preferred Table I B the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.9] to [0167.0.0.9]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.9.9] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contains and is listed as Table II the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 aminoacid sequence shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or one of 101-102 or the 478-481 sequence shown in capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.9] to [0172.0.0.9]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.9.9] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:8673 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:8673 sequence before use.

[0174.0.0.9] sees [0174.0.0.0]

[0175.0.9.9] for example, the sequence that has 80% homology at protein level and SEQ ID NO:8674 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQID NO:8674 sequence.

[0176.0.9.9] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the functional equivalent that obtains of one of polypeptide be listed as according to of the present invention as Table II the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide have essentially identical character and discern.

[0177.0.9.9] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the functional equivalent that obtains of nucleotide sequence be listed as according to of the present invention as Table II the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the polypeptide of the essentially identical character of polypeptide.

[0178.0.0.9] sees [0178.0.0.0]

[0179.0.9.9] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in) in introduce the replacement of one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleic acid molecule of homologue of protein sequence.Can by standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 introduce sudden change in the encoding sequence of sequence shown in capable.

[0180.0.0.9] to [0183.0.0.9]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.9.9] is employed to have as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleotide sequence homologous compound of sequence, perhaps come Table II the 5th or 7 row freely, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the homologous compound of nucleotide sequence of sequence also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.9.9] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 arbitrary sequence shown in capable in other Nucleotide of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.9.9] also preferred the inventive method comprise as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence identical.

[0187.0.9.9] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprise as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in encoding sequence identical.

[0188.0.9.9] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in and under the same conditions the polypeptide expressed activity compare and do not reduce substantially.In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence or by its homologue of forming.

[0189.0.9.9] as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the homologous compound of sequence, or deutero-as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or the homologue of 101-102 or the 478-481 sequence shown in capable also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.9] to [0191.0.0.9] sees that [0190.0.0.0] is to [0191.0.0.0]

[0191.1.9.9]-/-

[0192.0.0.9] to [0203.0.0.9] sees that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.9.9] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding is being listed as Table II the 5th or 7 of mature form at least, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given each fine chemicals in biological or its part, be vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol (89-92 or 482 row), alpha-tocopherol (93-95 or 472-475 are capable) particularly, 5,8-dimethyl tocol (96-100 or 476-477 are capable) and/or Gamma-Tocopherol (101-102 or 478-481 are capable) quantity increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 primer shown in capable or primer obtain amplification from cDNA library or genomic library nucleic acid molecule, and in biological or its part, give each fine chemicals, be vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol (89-92 or 482 row), alpha-tocopherol (93-95 or 472-475 are capable) particularly, the increase of 5,8-dimethyl tocol (96-100 or 476-477 are capable) and/or Gamma-Tocopherol (101-102 or 478-481 are capable) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in consensus sequence and in biological or its part, give each fine chemicals, be vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol (89-92 or 482 row), alpha-tocopherol (93-95 or 472-475 are capable) particularly, the nucleic acid molecule that 5,8-dimethyl tocol (96-100 or 476-477 are capable) and/or Gamma-Tocopherol (101-102 or 478-481 are capable) quantity increase;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol (89-92 or 482 row), alpha-tocopherol (93-95 or 472-475 are capable) particularly, the nucleic acid molecule that 5,8-dimethyl tocol (96-100 or 476-477 are capable) and/or Gamma-Tocopherol (101-102 or 478-481 are capable) quantity increase, described peptide coding such as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or the structural domain of 101-102 or the 478-481 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 nucleic acid molecule shown in capable or coding (optimized encoding is mature form at least) as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the 15nt at least of nucleic acid molecule of polypeptide, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as Table I A the 5th or 7 row, is respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention not by Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable is formed.In another embodiment, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as Table II A the 5th or 7 row, is respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 peptide sequence shown in capable.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 polypeptide shown in capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as Table II A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the protein of sequence.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in protein sequence at least 30% identical and with Table II A the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% is identical.

Thus, in another embodiment, (a) be different from as Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable by one or more Nucleotide to the nucleic acid molecule of (l).In one embodiment, nucleic acid molecule not by Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable is formed.In other embodiments, nucleic acid molecule of the present invention and Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as Table II B the 7th row, is respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 peptide sequence shown in capable.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 polypeptide shown in capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as Table II B the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the protein of sequence.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence form.In another embodiment, protein of the present invention and Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in protein sequence at least 30% identical and with Table II B the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% is identical.

[0205.0.0.9] to [0206.0.0.9]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.9.9] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of fertility phenol metabolism, fertility leukotriene metabolite, carotenoid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.9] to [0226.0.0.9]: see that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.9.9] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the capable sequence or derivatives thereof of mentioning of 478-481, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra for example other genes of vitamin-E precursor biosynthetic pathway of at least one tocopherol of expressing in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.9.9] therefore cultivated to cross simultaneously in another embodiment of the present invention and expressed coding and tocopherol metabolism (particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol are synthetic) at least one nucleic acid of related protein or the biology of a gene.

Other favourable nucleotide sequences that [0229.0.9.9] can express with the combination of sequence that present method is used and/or aforementioned biosynthesis gene are the sequence of other gene of coding tocopherol biosynthesis pathway, for example the enzyme of homogentisic acid phytyl transferring enzyme (HPT) or follow-up cyclisation of catalysis and methylation reaction, gama-tocopherol methyl transferase (γ-TMT), with the amylene transferring enzyme of isopentene group and the condensation of allyl group chain and those with the enzyme of isopentene chain and aryl condensation and other.These genes cause increasing essential vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol synthetic.

[0230.0.0.9] sees [0230.0.0.0]

[0231.0.9.9] is in another advantageous embodiment of the inventive method, the employed biology of present method is to have weakened degraded vitamin-E or its precursor 2 simultaneously, the protein of 3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol, especially those biologies of the expression rate by reducing corresponding gene.

[0232.0.0.9] to [0276.0.0.9]: see that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.9.9] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.Each fine chemicals of producing by this process can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing plant part or extract realization.

[0278.0.0.9] to [0282.0.0.9]: see that [0278.0.0.0] is to [0282.0.0.0]

[0283.0.9.9] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in protein, perhaps as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the antibody of polypeptide or its antigen part, it can utilize polypeptide of the present invention or its fragment (being polypeptide of the present invention) to produce by standard technique.Preferably specificity in conjunction with as Table II the 5th or 7 row, be respectively 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the monoclonal antibody of polypeptide.

[0284.0.0.9]: see [0284.0.0.0]

[0285.0.9.9] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence or by as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.9.9] in an advantageous embodiment, improved in the inventive method contain just like Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in consensus sequence or by the activity of its polypeptide of forming.In another embodiment, the present invention relates to contain just like Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in consensus sequence or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the polypeptide of consensus sequence (each row).

[0287.0.0.9] to [0290.0.0.9]: see that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.9.9] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as Table II A or IIB the 5th or 7 row, is respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 sequence shown in capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence form.

[0292.0.0.9] sees [0292.0.0.0]

[0293.0.9.9] the present invention relates to give in biology or its part that each fine chemicals increases and in one embodiment by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention.In one embodiment, described polypeptide have by one or more amino acid with as Table II A or IIB the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in other sequence of sequence phase region.In another embodiment, polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence form.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.

[0294.0.9.9] in one embodiment, the present invention relates to have as Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the polypeptide of activity of proteins, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence.

[0295.0.0.9] to [0297.0.0.9]: see that [0295.0.0.0] is to [0297.0.0.0]

The chemical of [0297.1.9.9] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the active polypeptide of polypeptide.

[0298.0.9.9] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the identical aminoacid sequence of sequence.

[0299.0.9.9] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 be listed as, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in one of nucleotide sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.9.9] therefore, described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with as Table II the 5th or 7 row, be respectively 89-92 or 482 capable and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence different.Therefore, this polypeptide contains and is listed as Table II A or IIB the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.9]see[0301.0.0.0]

The biologically-active moiety of [0302.0.9.9] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 aminoacid sequence shown in capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.9]: see [0303.0.0.0]

[0304.0.9.9] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.9] to [0306.0.0.9]: see that [0305.0.0.0] is to [0306.0.0.0]

[0306.1.9.9] preferably, compound is to comprise pure basically fine chemicals, it is vitamin-E, be alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol or vitamin-E precursor 2,3-dimethyl-5-phytyl chinol or recovery or isolating vitamin-E, be alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol or vitamin-E precursor 2, the composition of 3-dimethyl-5-phytyl chinol (particularly free form or protein bound form or film combining form).

[0307.0.0.9] to [0308.0.0.9]: see that [0307.0.0.0] is to [0308.0.0.0]

[0309.0.9.9] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 protein shown in capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 " the non-polypeptide of the present invention " or " other polypeptide " in capable is meant the aminoacid sequence with corresponding following proteins polypeptide, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, it is proteinic and shown in 68-71 and 450-459 are capable from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or capable " the non-polypeptide of the present invention " or " other polypeptide " of 478-481 do not give that each fine chemicals increases in biology or its part.

[0310.0.0.9] to [0334.0.0.9]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.9.9] confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or the expression of 101-102 or the 478-481 nucleotide sequence shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.9] to [0342.0.0.9]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.9.9] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.9] to [0361.0.0.9]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.9.9] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 polypeptide shown in capable, for example coding has protein such as Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the polypeptide of polypeptide active) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.In one embodiment, have as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide (for example have as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

[0363.0.0.9]: see [0363.0.0.0]

[0364.0.9.9] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---and for example naturally occurring as Table II the 3rd row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the promotor of polypeptide of the present invention be listed as with corresponding as Table I the 3rd, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the combination of protein coding sequence---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.9] to [0373.0.0.9]: see that [0365.0.0.0] is to [0373.0.0.0]

[0374.0.9.9] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the vitamin-E that produces in the inventive method or its precursor 2,3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol also can be from plant with free vitamin E or be bonded to compound or the isolated in form of part (moiety).By vitamin-E or its precursor 2 that this method produces, 3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.9] to [0376.0.0.9]: see that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.9.9] therefore the invention still further relates to the method according to this invention, separated the composition that contains vitamin-E that is produced or each fine chemicals that is produced thus.

[0378.0.9.9] can separate by this way and be higher than 50% by weight, favourable is higher than 60%, preferably be higher than 70%, especially preferably be higher than 80%, extremely preferably be higher than 90% the vitamin-E or its precursor 2 that produce in the method, 3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol.As required, subsequently can be with vitamin-E or its precursor 2 of resulting recovery, isolated or purified, 3-dimethyl-5-phytyl chinol, particularly, alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol (composition that for example comprises the former) be further purified, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.9.9] in one embodiment, vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol are the mixture that comprises one or more each fine chemicals.In one embodiment, each fine chemicals meaning here is meant vitamin-E, particularly alpha-tocopherol, 5,8-dimethyl tocol or Gamma-Tocopherol.In one embodiment, vitamin-E the meaning here is meant the mixture of each fine chemicals.

The vitamin-E that [0380.0.9.9] obtains in the method for the invention or its precursor 2, the suitable synthetic parent material of 3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises that separation produces comprises vitamin-E or its precursor 2, the composition of 3-dimethyl-5-phytyl chinol or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine that the inventive method produces.

[0381.0.0.9] to [0382.0.0.9]: see that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.9.9]：./.

[0384.0.0.9]: see [0384.0.0.0]

The fermented liquid that [0385.0.9.9] obtains with this kind approach, particularly contain and other compound, particularly other VITAMIN or carotenoid (as astaxanthin) or lipid acid blended vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, the fermented liquid of 5,8-dimethyl tocol and/or Gamma-Tocopherol, perhaps contain and comprise and other compound such as carotenoid blended vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol, alpha-tocopherol particularly, the fermented liquid of the microorganism of 5,8-dimethyl tocol and/or Gamma-Tocopherol or microorganism part (as plastid), dry matter content accounts for 7.5 to 25% of weight usually.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means that the concentration that can utilize sugar in the fermention medium during this period of time remains on or be reduced to 0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

Because vitamin-E is usually located at film or plastid, therefore in one embodiment, when by separation method such as centrifugal, filtration, decant, condense/flocculate or the combination of these methods from the fermented liquid kind completely or partially during the separating bio amount, avoiding cell is favourable by filtration.The biomass of doing directly can be added in the animal-feed, as long as the enough height and do not have toxic chemical of the concentration of vitamin-E.Consider the unstable of vitamin-E, drying conditions such as spraying drying or expansion drying are wanted gentle and will be avoided oxidation and suitable/anti-isomerization.Can add antioxidant, for example BHT, ethoxyquinoline or other antioxidant.Under the concentration of vitamin-E situation to be diluted, can use solvent extraction to separate, for example use alcohol, ether or other organic solvent (as methyl alcohol, ethanol, acetone, potassium hydroxide alcohol, phenol glycerine, liquefied phenol) or with acid or alkali such as trichoroacetic acid(TCA) or potassium hydroxide.Can find the multiple favorable method and the technology of separating VE in current state of the art.

[0386.0.9.9] therefore can also be further purified vitamin-E or its precursor 2 of generation, 3-dimethyl-5-phytyl chinol, particularly alpha-tocopherol, 5,8-dimethyl tocol and/or Gamma-Tocopherol.For this reason, the composition that will contain product, the lipid extracts fraction total or part that for example with an organic solvent obtains is as mentioned above carried out saponification (to remove triacylglycerol), distributes (distributing) (from more separating nonpolar epiphase the low phasic property derivative of polar) and separate (by for example open column chromatography or HPLC) between hexane/methanol, and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.9] to [0392.0.0.9]: see that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.9.9] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I, preferred IB the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 nucleic acid molecule shown in capable is hybridized nucleic acid molecule under lax stringent condition, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.9] to [0398.0.0.9]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.9.9] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving fine chemicals generation raising under these conditions.

Whether [00399.1.9.9] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect and depends on as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or the 478-481 polypeptide shown in capable or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.9] to [0416.0.0.9]: see that [0400.0.0.0] is to [0416.0.0.0]

[0417.0.9.9] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of vitamin-E biosynthetic pathway is had resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block vitamin-E in the described biology (particularly each fine chemicals) synthetic inhibitor.

Biological antioxidant is coerced, the particularly damage of singlet oxygen because vitamin-E can be protected, and the level that therefore increases each fine chemicals can the anti-weedicide that causes producing oxygenated compound (for example singlet oxygen) of protective plant.For example, the inhibition to a kind of important enzyme proporphyrinogen oxidase (Protox) in chlorophyll and the protoheme biosynthesizing causes the loss of Chlorophylls and Carotenoids and causes producing the seepage film; The destruction of film is owing to the generation (weedicide for other typical photosynthetic inhibitor that becomes second nature also has report) of free oxygen free radical.

Therefore, in one embodiment, the increase of each fine chemicals level is used for the anti-weedicide that destroys film (because generation of free oxygen free radical) of protective plant.

Producing the inhibitor of oxidative stress or the example of weedicide is aryl triazoles (aryl triazion), for example sulfentrazone, fluorine ketazolam grass; Perhaps diphenyl ether, for example acifluorfen, lactofen or Oxyfluorfen; Perhaps N-phenylphthalimide, for example imide phenoxy acetic acid Huo Fu oxazinone; Substituted urea class, for example fluometuron, Metribuzin, Diuron Tech or methoxydiuron; Triazines, for example G-30027, prometryn, ametryn, sencorex, prometon, simazine or six piperazines are together; Perhaps uracil, for example bromacil or terbacil.

[0418.0.0.9] to [0423.0.0.9]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.9.9] therefore, nucleic acid of the present invention or the inventive method nucleic acid molecule used therefor, polypeptide of the present invention or the used polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other carotenoid, VITAMIN or lipid acid.In one embodiment, in the methods of the invention, the vitamin-E that is produced is used to protect lipid acid to avoid oxidation, for example in another step with its with purified form or only the isolating form of part join in the composition that contains lipid acid.

Therefore, nucleic acid of the present invention or the inventive method nucleic acid molecule used therefor or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention or the used polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0424.1.9.9] the present invention relates to the purposes that antagonist of the present invention, plant of the present invention or its part, microorganism of the present invention or host cell or its part are used for production cosmetic compositions or pharmaceutical composition in other embodiments.This composition has anti-oxidant activity, the light protection is active, and it can be used for protection, treats or cure above-mentioned disease (for example hypercholesterolemia or cardiovascular disorder, some cancer and cataract form) or be used as immunostimulant.

Vitamin-E can be used as other color sensitivity or oxygen sensitive compound, for example stablizer of lipid acid, particularly unsaturated fatty acids.

[0425.0.0.9] to [0434.0.0.9]: see that [0425.0.0.0] is to [0434.0.0.0]

[0435.0.9.9] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.9.9] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement green algae (Spongiococcum sp (for example Spongiococcumexentricum) for example, Chlorella certain (Chlorella sp.), haematococcus, Phaeodactylum tricornutum, volvox or Dunaliella salina belong to), cytoalgae PCC 6803, exhibition leaf sword-like leave moss, yeast belong, genus mortierella, the mutagenesis in vivo of Escherichia and above-mentioned other genus is beneficial to produce vitamin-E.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coliand Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces the expection fine chemicals by screening can screen positive catastrophic event.

[0436.1.0.9] sees [0436.1.0.0]

[0437.0.9.9] to [0445.0.9.9]: see that [0437.0.5.5] is to [0445.0.5.5]

[0445.1.9.9] cytoalgae PCC 6803 is single celled non-fixed nitrogen cyanobacterias; this cyanobacteria comprehensive gene studies (Churin etc. have been carried out; (1995) J Bacteriol 177:3337-3343), it can easily be transformed (Williams (1988) Methods Enzymol 167:766-778) and be had very active homologous recombination potential.Bacterial strain PCC 6803 is isolating from the fresh water of California, USA as Aphanocapsa N-1 in nineteen sixty-eight by R.Kunisawa, can pass through " Pasteur Culture Collection of AxenicCyanobacterial Strains " (PCC) (Unit é de Physiologie Microbienne now, Paris France) obtains.The full gene group sequence of cytoalgae PCC 6803 begins to announce (Kaneko etc., (1995) DNA Research 2:153-166 from nineteen ninety-five; Kaneko etc., (1995) DNA Research 2:191-198; Kaneko etc., (1996) DNA Research3:109-136; Kaneko etc., (1996) DNA Research 3:185-209; Kaneko and Tabata (1997) Plant Cell Physiol 38:1171-1176; Kotani and Tabata (1998) Annu Rev Plant Physiol 49:151-171) and on Internet announce (http://www.kazusa.or.jp/cyano/cyano.html) with title " CyanoBase ".The effective expression system description that is used for cytoalgae 6803 is in document (Mermet-Bouvier etc., (1993) Curr Microbiol 27:323-327; Mermet-Bouvier and Chauvat (1993) CurrMicrobiol 28:145-148; Murphy and Stevens (1992) Appl Environ Microbiol58:1650-1655; Takeshima etc., (1994) Proc Natl Acad Sci USA 91:9685-9689; Xiaoqiang etc., (1997) Appl Environ Microbiol 63:4971-4975; Ren etc., (1998) FEMS Microbiol Lett 158:127-132).

The cultivation of [0445.2.9.9] cytoalgae

Cytoalgae PCC 6803 cells are normal from nourishing one's nature cultivation in the BG11 substratum.Their diameter is 2.3-2.5 μ m.For example, can use cyanobacteria cytoalgae PCC 6803 bacterial strains of tolerance glucose, different oxygen growth that promptly it can also be in the dark the weak blue illumination of several minutes (every day).Culture condition is by Anderson and McIntosh exploitation (Anderson and McIntosh (1991) J Bacteriol173:2761-2767) and be called the different oxide growth of photoactivation (LAHG).This makes can cultivate these cyanobacterias, and does not have the light compositing that continues and therefore also do not have oxygen to produce.

BG 11 substratum that are used for cytoalgae

Stock solution, 100x BG11:

NaNO ₃??????????1.76M＝149.58g

MgSO ₄x?7H ₂O????30.4mM＝7.49g

CaCl ₂x?2H ₂O????24.5mM＝3.6g

Citric acid 3.12mM=0.6g

Na?EDTA?pH?8???0.279mM＝0.104g

With the substance dissolves of weighing in 900ml H ₂O also complements to 1000ml with 100ml trace-metal mixing stock solution (1000x).The solution that so obtains can be used as stock solution.

Trace-metal mixing stock solution, 1000x:

H ₃BO ₃????????????46.3mM＝2.86g/l

MnCl ₂x?4H ₂O??????4.15mM＝1.81g/l

ZnSO ₄x?7H ₂O??????0.77mM＝0.222g/l

Na ₂MoO ₄x?2H ₂O????1.61mM＝0.39g/l

CuSO ₄x?5H ₂O??????0.32mM＝0.079g/l

Co(NO ₃) ₂x?6H ₂O????0.17mM＝0.0494g/l

Following solution is that 1 liter of BG11 culture solution of preparation is required:

1.10ml stock solution 100x BG 11

2.1ml?Na ₂CO ₃(189mM)

3.5ml?TES(1M，pH?8)

4.1ml?K ₂PO ₄(175mM)

Although but solution 2 and 3 filtration sterilizations, solution 4 must autoclaving.To BG11 culture solution autoclaving completely before use, mix with the previous 1ml ferric ammonium citrate (6mg/ml) of filtration sterilization then.Never will be with the ferric ammonium citrate autoclaving.For agar plate, add 1.5% (w/v) bacto agar in every liter of BG11 substratum.

Amplification and the clone of [0445.3.9.9] cytoalgae PCC 6803DNA

The method (Howitt CA (1996) BioTechniques21:32-34) of using Crispin A.Howitt is by polymerase chain reaction (PCR) DNA amplification from cytoalgae PCC 6803.

The generation of tocopherol among [0445.4.9.9] cytoalgae PCC 6803

Cultivate on the BG-11km nutrient agar each independently the cell of cytoalgae PCC 6803 bacterial strains and the wild-type cell (not containing on the BG11 nutrient agar of kantlex) of non-conversion can be used for being seeded to liquid nutrient medium.For this reason, mutant or wild-type cytoalgae PCC 6803 cells are transferred to the 10ml liquid nutrient medium from flat board.At 28 ℃ and 30 μ mol photon * (m ²* s) ^-1Under (30 μ E) condition, these cultures were cultivated about 3 days.At the OD that measures each culture ₇₃₀Afterwards, by using BG-11 (for wild-type) or BG-11km (for mutant) appropriately to dilute OD with all cultures ₇₃₀Consistent.The culture (mutant and wild-type contrast) of these cell density unanimities is seeded in 3 parts of substratum.Therefore, use can be carried out biochemical analysis for three parts of independent grown culture under mutant and each situation of corresponding wild-type.It is OD that culture is cultivated until optical density(OD) ₇₃₀=0.3.

By in the Eppendorf desk centrifuge, removing cell culture mediums centrifugal twice with 14000 rev/mins.Then, in 100% methyl alcohol, on the Eppendorf wobbler, shake 15 minutes (twice, the supernatant that obtains is mixed) with cytoclasis and extract tocopherol or vitamin-E in 30 ℃, 1000 rev/mins.

For fear of oxidation, after extraction, the extract that obtains is analyzed immediately by means of Waters Allience 2690HPLC system.(ProntoSil 200-3-C30 Bischoff) uses 100% methyl alcohol to separate tocopherol and vitamin-E, and identifies by standard method (Merck) on reversed-phase column.The fluorescence of material (excitation wavelength 295nm, emission wavelength 320nm) can detect by Jasco FP 920 fluorimetric detectors, and this fluorescence can be used as detection system.

[0446.0.0.9] to [0450.0.0.9]: see that [0446.0.0.0] is to [0450.0.0.0]

[0451.0.0.9]: see [0451.0.5.5]

[0452.0.0.9] to [0454.0.0.9]: see that [0452.0.0.0] is to [0454.0.0.0]

The feature of [0455.0.9.9] transgenic plant

Content for tocopherol and vitamin-E in the leaf of the conversion plant (Arabidopis thaliana, colea and tobacco) that confirms that polypeptide expression described herein can influence the biosynthesizing of vitamin-E in the transgenic plant, analyzed to contain described construct and the seed., transgenic plant are grown in the greenhouse for this reason, and identified the plant of expressing the gene of code book invention polypeptide or the used polypeptide of the inventive method in the Northern level.Measure the leaf of these plants and tocopherol content or content of vitamin E in the seed.Generally speaking, compare with non-conversion plant, tocopherol content has improved.

[0456.0.0.9]: see [0456.0.0.0]

[0457.0.9.9] embodiment 9: vitamin-E or its precursor 2, the purifying of 3-dimethyl-5-phytyl chinol

[0458.0.9.9] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

Described by using (1997, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/massspectrometric method], Lipide 33:343-353) standard method of analysis GC, GC-MS or TLC can clearly detect vitamin-E to the biology analysis of recombinating, breast alpha-tocopherol, 5,8-dimethyl tocol or Gamma-Tocopherol or its precursor 2, the existence of 3-dimethyl-5-phytyl chinol.

It is biological to use the following step to analyze, as the total vitamin-E that produces in the inventive method used yeast: by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

[0459.0.9.9] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified vitamin-E, for example alpha-tocopherol, 5,8-dimethyl tocol or Gamma-Tocopherol or its precursor 2,3-dimethyl-5-phytyl chinol.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.9.9] sees [0460.0.0.0]

[0461.0.9.9] embodiment 10: clone SEQ ID NO:8673 is used for expressing plant

[0462.0.0.9]: see [0462.0.0.0]

[0463.0.9.9] passes through pcr amplification SEQ ID NO:8673 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.9] to [0466.0.0.9]: see that [0464.0.0.0] is to [0466.0.0.0]

[0467.0.9.9] selects following primer sequence for genes of SEQ ID NO:8673:

I) forward primer (SEQ ID NO:8677)

atggggaaga?gagtatacga?tcca

Ii) reverse primer (SEQ ID NO:8678)

tcactccagc?ttaaacatgg?cgg

[0468.0.0.9] to [0479.0.0.9]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.9.9]: embodiment 11: express the generation of the transgenic plant of SEQ ID NO:8673

[0481.0.0.9] to [0513.0.0.9]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.9.9] selects as another kind of, as described (Deli, J.﹠amp; Molnar, P., Paprikacarotenoids:Analysis, isolation, structure elucidation.Curr.Org.Chem.6,1197-1219 (2004) or Fraser, P.D., Pinto, M.E., Holloway, D.E.﹠amp; Bramley, P.M.Technical advance:application of high-performance liquidchromatography with photodiode array detection to the metabolicprofiling of plant isoprenoids.Plant J.24,551-558 (2000)) advantageously detects vitamin-E, for example alpha-tocopherol, 5,8-dimethyl tocol or Gamma-Tocopherol or its precursor 2,3-dimethyl-5-phytyl chinol.

The different plants of being analyzed the results are shown in following table:

Table 1

ORF	Metabolite	Method	??Min	??Max
					YPL268W	2,3-dimethyl-5-phytyl chinol	??LC	??1.44	??1.51
YFL013C	2,3-dimethyl-5-phytyl chinol	??LC	??1.97	??5.40
					b1829	2,3-dimethyl-5-phytyl chinol	??LC	??1.48	??3.83
b1827	2,3-dimethyl-5-phytyl chinol	??LC	??1.76	??1.91
					YFL019C	2,3-dimethyl-5-phytyl chinol	??LC	??1.97	??5.40
b2699	Alpha-tocopherol	??GC	??1.51	??2.96
					b1829	Alpha-tocopherol	??LC	??1.39	??1.90
b0112	Alpha-tocopherol	??LC	??1.43	??1.63
					b0161	Alpha-tocopherol	??GC	??1.50	??2.38
b0970	Alpha-tocopherol	??LC	??1.47	??1.54
					b3160	Alpha-tocopherol	??GC	??1.47	??1.91
b4063	Alpha-tocopherol	??LC	??1.16	??1.47
					b1827	5,8-dimethyl tocol	??LC	??1.34	??2.16
b0986	γ+5,8-dimethyl tocol	??LC	??1.35	??1.61
					b0175	γ+5,8-dimethyl tocol	??LC	??l.34	??1.40
b0785	γ+5,8-dimethyl tocol	??LC	??1.34	??1.85
					b3938	γ+5,8-dimethyl tocol	??LC	??1.50	??2.20
YFL053W	γ+5,8-dimethyl tocol	??LC	??1.45	??2.23

??ORF	Metabolite	Method	??Min	??Max
					??b1829	γ+5,8-dimethyl tocol	??LC	??1.40	??4.25

[0515.0.9.9] to [0552.0.0.9]: see that [0515.0.0.0] to [0552.0.0.0] comprises [0530.1.0.0] to [0530.6.0.0] and [0552.1.0.0] and [0552.2.0.0]

[0553.0.9.9]

1. produce vitamin-E or its precursor 2, the method for 3-dimethyl-5-phytyl chinol, it comprises:

(a) in non-human being or its one or more parts, improve or produce one or more as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 are capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, produce vitamin-E or its precursor 2, cultivate this biology under the condition of 3-dimethyl-5-phytyl chinol.

2. produce vitamin-E or its precursor 2, the method for 3-dimethyl-5-phytyl chinol is included in to improve in biology or its part or produce and expresses at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide or its fragment, described nucleic acid molecule is given vitamin-E or its precursor 2 in biology or its part, and 3-dimethyl-5-phytyl chinol quantity increases;

B) contain just like Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or the nucleic acid molecule of 101-102 or the 478-481 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part vitamin-E or its precursor 2, the nucleic acid molecule that 3-dimethyl-5-phytyl chinol quantity increases;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in vitamin-E or its precursor 2, the nucleic acid molecule that 3-dimethyl-5-phytyl chinol quantity increases;

E) under stringent hybridization condition, hybridize and give vitamin-E or its precursor 2 in biology or its part, the nucleic acid molecule that 3-dimethyl-5-phytyl chinol quantity increases with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give vitamin-E or its precursor 2 in biology or its part, 3-dimethyl-5-phytyl chinol quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give vitamin-E or its precursor 2 in biology or its part, 3-dimethyl-5-phytyl chinol quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in consensus sequence polypeptide and give biology or its part in vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol quantity increases; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give vitamin-E or its precursor 2 in biology or its part, 3-dimethyl-5-phytyl chinol quantity increases, and wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt; Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, wherein 2,3-dimethyl-5-phytyl chinol is separated.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded vitamin-E or its precursor 2 of selected mutation biology or the generation of its part, 3-dimethyl-5-phytyl chinol.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table II B the 7th row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in polypeptide or its fragment, described nucleic acid molecule is given vitamin-E or its precursor 2 in biology or its part, and 3-dimethyl-5-phytyl chinol quantity increases;

B) contain just like Table I the 5th or 7 row, be respectively 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 is capable, preferred Table I B the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or the nucleic acid molecule of 101-102 or the 478-481 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part vitamin-E or its precursor 2, the nucleic acid molecule that 3-dimethyl-5-phytyl chinol quantity increases;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in vitamin-E or its precursor 2, the nucleic acid molecule that 3-dimethyl-5-phytyl chinol quantity increases;

E) under stringent hybridization condition, hybridize and give vitamin-E or its precursor 2 in biology or its part, the nucleic acid molecule that 3-dimethyl-5-phytyl chinol quantity increases with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give vitamin-E or its precursor 2 in biology or its part, 3-dimethyl-5-phytyl chinol quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give vitamin-E or its precursor 2 in biology or its part, 3-dimethyl-5-phytyl chinol quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in consensus sequence polypeptide and give biology or its part in vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol quantity increases; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give vitamin-E or its precursor 2 in biology or its part, 3-dimethyl-5-phytyl chinol quantity increases, and wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt; Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, 89-92 or 482 row and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding as claim 6 described in, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 89-92 or 482 capable and/or 93-95 or 472-475 is capable and/or 96-100 or 476-477 is capable and/or 101-102 or 478-481 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in vitamin-E or its precursor 2,3-dimethyl-5-phytyl chinol quantity increases, described method comprises:

(a) will express codedly and give vitamin-E or its precursor 2 in biology or its part by the nucleic acid molecule of claim 6, cell, tissue, plant or the microorganism of the polypeptide that 3-dimethyl-5-phytyl chinol quantity increases and candidate compound or the sample that comprises multiple compound contact under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps cultivate or keep vitamin-E or its precursor 2 in the substratum of cell, tissue, plant or microorganism, 3-dimethyl-5-phytyl chinol level or expression of polypeptides level; With

(c) vitamin-E or its precursor 2 by measuring, 3-dimethyl-5-phytyl chinol level or expression of polypeptides level and standard vitamin-E or its precursor 2 when described candidate compound or the sample that comprises described multiple compound lack, measured, 3-dimethyl-5-phytyl chinol or expression of polypeptides level are relatively identified agonist or antagonist; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and give vitamin-E or its precursor 2 in plant or the microorganism, 3-dimethyl-5-phytyl chinol produces the method for the compound that improves, and said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in vitamin-E or its precursor 2, the polypeptide that 3-dimethyl-5-phytyl chinol quantity increases and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and the nucleic acid molecule that can be provided at described read-out system of permission and claim 6 is coded and give vitamin-E or its precursor 2 in biology or its part, response compound and described polypeptide bonded detectable signal under the condition of the expression of polypeptides that 3-dimethyl-5-phytyl chinol quantity increases; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and give vitamin-E or its precursor 2 in the cell, 3-dimethyl-5-phytyl chinol produces the method for the gene product that improves, and said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain gives vitamin-E or its precursor 2 after being coded in expression, the candidate gene of the gene product that 3-dimethyl-5-phytyl chinol increases;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported suitable vitamin-E or its precursor 2, the host cell of 3-dimethyl-5-phytyl chinol of producing;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) vitamin-E or its precursor 2 in the analysis host cell, 3-dimethyl-5-phytyl chinol level; With

(f) identify nucleic acid molecule and gene product thereof, compare with wild-type that it gives vitamin-E or its precursor 2 in the host cell after expressing, 3-dimethyl-5-phytyl chinol level improves.

20. identify and give vitamin-E or its precursor 2 in the cell, 3-dimethyl-5-phytyl chinol produces the method for the gene product that improves, and said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain gives vitamin-E or its precursor 2 in biology or its part after being coded in expression, the candidate gene of the gene product that 3-dimethyl-5-phytyl chinol quantity or level improve, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported suitable vitamin-E or its precursor 2, the host cell of 3-dimethyl-5-phytyl chinol of producing;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) vitamin-E or its precursor 2 in the analysis host living beings, 3-dimethyl-5-phytyl chinol level; With

(e) identify nucleic acid molecule and gene product thereof, compare with wild-type that it gives vitamin-E or its precursor 2 in the host cell after expressing, 3-dimethyl-5-phytyl chinol level improves.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used for identifying gives vitamin-E or its precursor 2, the nucleic acid molecule that 3-dimethyl-5-phytyl chinol increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying and can gives regulation and control vitamin-E or its precursor 2, the compound of 3-dimethyl-5-phytyl chinol level at biology.

25. makeup, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the polypeptide of the nucleic acid molecule of any one described method, claim 6, claim 14, claim 7 among the claim 1-5, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein vitamin-E be respectively α-, β-or Gamma-Tocopherol.

27. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make, and plant is anti-oxidant coerces.

28. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make the anti-weedicide that causes oxidative stress of plant.

29. the purposes of the results material of the agonist of identifying according to claim 17, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12, it is used to produce makeup or pharmaceutical composition.

[0554.0.0.9] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.10] sees [0000.0.0.1]

[0001.0.0.10] sees [0001.0.0.0]

[0002.0.10.10] carotenoid is in redness, yellow and the orange pigment that is distributed widely in day right boundary.Though the photosynthetic maincenter in plant, plumage, crustaceans and mary bush petal identifies certain kinds carotene, they are abundant especially in yellowish-orange fruits and vegetables and blackish green leaf vegetables.Identified so far more than 700 kinds of naturally occurring carotenoid in, have in 50 can be absorbed by the body and metabolism.Up to the present, only identify 14 kinds of carotenoid in the human serum kind.

In animal, some carotenoid (particularly β-Hu Luobusu) can be used as the precursor vitamin A in the diet, and some of them can be brought into play the fat-soluble antioxidant effect.In plant, carotene can be used as the reactive photosynthetical system of antioxidant protection height, and can be used as the fill-in light pigment and play a role.In experiment in vitro, show Lyeopene, alpha-carotene, zeaxanthin, xenthophylls and cryptoxanthin cancellation singlet oxygen and suppressed lipid peroxidation.The oxidative metabolites of xenthophylls, zeaxanthin and Lyeopene separate and evaluation provides the direct evidence of the antioxygenation of these carotenoid.

Carotenoid is 40-carbon (C ₄₀) terpenoid, comprise 8 isoprene (C that connect together usually ₅) unit.The unitary branch subcenter that is connected is a reversible." ketone group carotenoid " is the generic term of carotenoid pigment, and ketocarotinoids comprises ketone group at the ionene loop section of molecule, and " hydroxy kind carotene " is meant the carotenoid pigment that comprises hydroxyl in the ionene ring.To use popular name and abbreviation in the whole disclosure, half systematic naming method that IUPAC recommends provides in the parantheses after mentioning popular name first usually.

Carotenoid is from 5 carbon atom metabolic precursor thereof-isopentenylpyrophosphate (IPP)-synthetic.Having two known biosynthetic pathways aspect the formation of IPP (general isoprene unit) at least.Article one, approach starts from that (mevalonic acid that 3-hydroxy-3-methyl glutaryl-CoA) forms, mevalonic acid is self to be transformed into isopentenylpyrophosphate (IPP) via HMG-CoA from acetyl-CoA.Afterwards, two Mang ox geranylpyrophosphates (GGPP) molecule condensation each other produces colourless phytoene, and it is initial carotenoid.Research has shown also and exists the alternative IPP that does not rely on mevalonic acid to form approach that the initial characteristic of this approach is present in the plastid of several eubacteriums, green alga and higher plant.In this alternative route first reaction is that the transketolase type condensation reaction of pyruvate salt and D-glyceraldehyde-3-phosphate obtains intermediate 1-deoxy-D-xylulose-5-phosphoric acid (DXP).

By the reaction of a series of desaturations, phytoene be converted into phytofluene ,-carotene, neurosporene, and form Lyeopene at last.Subsequently, Lyeopene is converted into β-Hu Luobusu by cyclization, and it comprises 2 β-ionene ring.Ketone group group and/or oh group are imported in each ring of β-Hu Luobusu, thus accumulated angle flavine, zeaxanthin, astaxanthin.Hydroxylase has shown and canthaxanthin can be converted into astaxanthin.Equally, ketolase has shown and zeaxanthin can be converted into astaxanthin.Ketolase can also be converted into canthaxanthin with β-Hu Luobusu, and hydroxylase can be converted into zeaxanthin with β-Hu Luobusu.

The carotenoid absorb light is in the 400-500nm zone of visible spectrum.This physical properties gives pigment distinctive redness/yellow.Usually put upside down in molecule central authorities by the conjugated backbone that isoprene unit is formed, give symmetry.The change of how much conformations causes existing many cis and trans-isomer(ide) around two keys.Also there are hydroxylation, oxidation, hydrogenation or contain the derivative of ring.Hydro carbons carotenoid can classify as carotene, and oxygen containing those hydro carbons carotenoid are known as xenthophylls.

In animal, carotenoid be by means of the fat in the diet from little intestinal absorption and mix chylomicron and transport into serum.Soluble selectivity distribution, biologic activity and the precursor vitamin A potential in organ-tissue of the different structure feature that carotenoid has, perhaps vitamin A transforms in the body.Since hydrophobic characteristic, the lipid part correlation of carotenoid and human tissue, cell and film.Generally speaking, the carotenoid of 80-85% is distributed in fatty tissue, is found in liver, muscle, suprarenal gland and reproductive organ on a small quantity.About 1% carotenoid is circulated in the serum with high-density and low-density lipoprotein.Serum-concentration is suitable constant and slowly changes in the low absorption cycle.For Lyeopene ,-carotene ,-carotene, xenthophylls and zeaxanthin, estimated half life, be estimated as 11-14 days.Announced the evidence that has more than one body storage ponds.Main serum carotenoid is-carotene ,-carotene, xenthophylls, zeaxanthin, Lyeopene and cryptoxanthin.Also there are Polyenes such as phytoene and phytofluene in a small amount.

The human serum level reflects in the Different Culture and the mode of life between the Different Culture is selected and food habits.Approximately only 15 kinds are circulated in the blood with HDL and LDL.Receptivity that variation can not wait owing to different absorptions, to some carotenoid and different metabolic rates and organize uptake rate.The serum level that reduces is followed use, smoking and the exposure duration generation of prolongation UV line of ethanol consumption, oral contraceptive.

-carotene ,-carotene and-cryptoxanthin is converted into Vogan-Neu or vitamin A by 15-15 '-b-carotenoid dioxygenase in small intestine and liver.Vogan-Neu forms and shows as Steady-State Control in this kind body, thereby makes Vogan-Neu transform the philtrum that only limits to have enough vitamin A.

[0003.0.10.10] fixed β-Hu Luobusu makes it become the part of different antioxidant systems of defense in the human body in the effect of harmful free radical of cancellation singlet oxygen and intercepting and reactive oxygen species.Reactive oxygen species has related to the generation of numerous disease, comprises that ischemic heart disease, various cancer, cataract and macula form.Because the conjugated polyene of β-Hu Luobusu is partly given its antioxidant ability and whole carotenoid all has this constitutional features, so the effect of other carotenoid of evaluation in the disease of prevention free radical mediated pointed in research work.Really, experiment in vitro is verified Lyeopene, alpha-carotene, zeaxanthin, xenthophylls and cryptoxanthin cancellation singlet oxygen also suppresses lipid peroxidation.The direct evidence of separating and identify the antioxygenation that these carotenoid can be provided of xenthophylls, zeaxanthin and Lyeopene oxidative metabolites.

Except the antioxidant ability, other biological action of carotenoid comprises the active and external gap junction communication of enhancing immunity, reduces or suppress the ability of mutagenesis and vitro inhibition cell transformation.

Diet absorption of yellowish-orange fruit and blackish green leaf vegetables and the negative correlation between multiple cancer (especially mouth, pharynx, larynx, esophagus, lung, stomach, uterine cervix and the bladder) incidence have been determined in many epidemiological studies.Though a large amount of protectiveness compounds can produce this kind observations, have been noted that all to have carotenoid simultaneously in these food.Because nutritionist and medical professional recognize the existence of a large amount of inhomogeneity carotene in the food at present, so it is become research direction at effect and biological impact aspect healthy.

Xenthophylls is present in retina.It protects the visual cell to avoid the injury of the oxyradical of light generation, and therefore has keying action aspect carrying out property of the prevention macular degeneration.But xenthophylls has the gap junction communication between chemoprophylaxis activity inducing cell, and suppresses lipid peroxidation external more effectively than β-Hu Luobusu, alpha-carotene and Lyeopene.High-caliber xenthophylls and lung cancer negative correlation in the serum have been proved.

Except xenthophylls, zeaxanthin is present in the retina and gives the provide protection of prevention maculopathy.Zeaxanthin also is prevalent in ovary and adipocyte tissue.This xenthophylls does not have the provitamin A activity.

Ethanol consumption has shown influences lipid peroxidation.After alcohol was taken in, though the concentration of these xenthophylls reduces, content was higher in blood plasma for dehydrogenation xenthophylls (oxidation by-product of xenthophylls and zeaxanthin).Therefore xenthophylls and zeaxanthin may have the provide protection of anti-LDL oxidation.

The alltrans isomer of Lyeopene generally all carries out in serum quantitatively, although total the signal of 9-, 13-and 15-cis-isomeride also be detectable and occupy 50% of Lyeopene.In the external experiment of carrying out, Lyeopene is than alpha-carotene, β-Hu Luobusu, zeaxanthin, xenthophylls and cryptoxanthin cancellation singlet oxygen more effectively.Lyeopene is induced gap junction communication, suppresses lipid peroxidation and shows the chemoproection activity.The serum level of Lyeopene and pancreas and Cervical cancer risk present negative correlation.This carotenoid has obtained identifying in Tiroidina, kidney, suprarenal gland, spleen, liver, heart, testis and pancreas.Lyeopene does not transform into Vogan-Neu in vivo.

β-cryptoxanthin can the cancellation singlet oxygen.β-cryptoxanthin is used to the butter that dyes.β-cryptoxanthin presents the provitamin A activity.

Because the alltrans isomer of this carotenoid has high vitamin A activity, so it is the main source of diet retinoid.The trans β-Hu Luobusu of a part can provide two molecule Vitamin A1 aldehydes in theory in vivo.In carotenoid spectrum, also observe the signal of the 13-of β-Hu Luobusu and 15-cis-isomeride and account for total β-Hu Luobusu in the serum 10% or still less.β-Hu Luobusu cancellation singlet oxygen is induced gap junction communication and is suppressed lipid peroxidation.The high serum level of β-Hu Luobusu is relevant with the low incidence of cancer in mouth, lung, breast, uterine cervix, skin and the stomach.At Tiroidina, kidney, spleen, liver, heart, pancreas, fat, ovary and suprarenal gland with identified β-Hu Luobusu.

Alpha-carotene is similar to β-Hu Luobusu aspect its biologic activity, but cancellation singlet oxygen more effectively.Alpha-carotene improves gap junction communication in vivo, stops lipid peroxidation and suppress carcinogens ground to form and take in.High serum level is low dangerous relevant with lung cancer.Though only have half provitamin A potential of β-Hu Luobusu, alpha-carotene also can recover normal cell growth and differentiation.Serum level usually total β-Hu Luobusu numerical value 10 and 20% between.

Alpha-carotene, β-Hu Luobusu and β-cryptoxanthin can change into vitamin A in enteron aisle and liver.Vitamin A is essential for immune response and also participates in other defense reaction at contagium.Even so, in many individualities, this conversion is very slow and is invalid, particularly for the elderly.More known individual right and wrong or low responders are because they can not be converted into vitamin A with β-Hu Luobusu with the expection ratio.A large amount of factors can suppress β-Hu Luobusu and be converted into vitamin A.The major cause that so many American suffers from the A situation that is deficient in vitamin is usually excessive drinking.Enterozoa may be a factor.And, need the prescription drug of liver metabolism will reduce that β-Hu Luobusu is converted into Vogan-Neu in the liver arbitrarily.Diabetic subject and the individuality of suffering from hypothyroidism or being in the hypothyroidism edge might be the low responders.

[0004.0.10.10] in plant, approximately 80-90% is present in the green leaves plant, and for example the carotenoid of cabbage, wild cabbage, spinach and brussels sprouts is xenthophylls, and 10-20% is a carotene.On the contrary, yellow and orange vegetables (comprising Radix Dauci Sativae, sweet potato and pumpkin) mainly contain carotene.In the micro-wave cooking process, destroy the xenthophylls of as many as 60% in these foods and 15% carotene.In xenthophylls, xanthophyll demonstrates the most stable.

Xenthophylls is present in mango, pawpaw, orange, Kiwifruit, peach, pumpkin, pea, lima bean, string bean, cabbage, brussels sprouts, rape, wild cabbage, lettuce, plum, pumpkin, sweet potato and honeydew melon.Commercial materials comes from the extract of mary bush petal.Xenthophylls does not have the provitamin A activity.

The diet source of zeaxanthin comprises peach, pumpkin, apricot, citrus, pawpaw, plum, pumpkin, mango, kale, Kiwifruit, lettuce, honeydew melon and Semen Maydis.

The redness of the skin of fruits and vegetables such as tomato, pink colour natsudaidai, red grape, watermelon and red guavas is because Lyeopene.Other diet source comprises pawpaw and apricot.

β-cryptoxanthin is present in citrus, mango, pawpaw, muskmelon, peach, plum, pumpkin.

The diet source of β-Hu Luobusu comprises mango, muskmelon, Radix Dauci Sativae, pumpkin, pawpaw, peach, plum, pumpkin, sweet potato, apricot, Btassica, Kidney bean, mung bean, cabbage, brussels sprouts, kale, Kiwifruit, lettuce, pea, spinach, tomato, pink colour natsudaidai, honeydew melon and citrus.

The diet source of alpha-carotene comprises sweet potato, apricot, pumpkin, muskmelon, mung bean, Kidney bean, cabbage, brussels sprouts, Btassica, kale, Kiwifruit, lettuce, pea, spinach, plum, peach, mango, pawpaw, pumpkin and Radix Dauci Sativae.

[0005.0.10.10] some carotenoid are present in the various marine animal bodies especially, comprise fish such as salmon and porgy and crustaceans such as crab, lobster and shrimp.Because animal usually can not biosynthesizing carotenoid, when they take in microorganism or plant, could obtain those and be present in wherein carotenoid so have only.Compare with those materials that obtain by methodology of organic synthesis, by biogenetic derivation for example the carotenoid supplied with of crustaceans, yeast and green alga for example xenthophylls, astaxanthin since the extracting method of its low yield and costliness be restricted.Yet common synthetic method can produce and be considered to unacceptable byproduct.Therefore a kind of carotenoid, particularly xenthophylls of relatively inexpensive source is found in expectation, as the fodder additives in the aquaculture and as valuable chemical in other industrial use and the diet.The source of xenthophylls comprises krill, Fife's yeast belong (Phaffia) zymic cultured products, the cultured products of green alga Haematocoocus Pluvialls (Haematococcus pluvialis) and the product that obtains by methodology of organic synthesis in the crustaceans such as the South Pole.Yet, when using crustaceans such as krill etc., in results and leaching process, need extensive work with expense so that from pollutent such as lipid etc., separate xenthophylls.Yet,,, need a large amount of expenses to be used for the enrichment and the extraction of astaxanthin because yeast has hard cell walls and only produces xenthophylls with low yield for Fife's yeast belong zymic cultured products.A kind of method that improves the productivity of some xenthophylls products in the biosystem is to use gene engineering.

[0006.0.10.10] in many plants, Lyeopene is the tapping point in the carotenoid biosynthesizing.Therefore, the Lyeopene of some plants is made into β-Hu Luobusu and zeaxanthin, and usually is zeaxanthin two glucosides, and the remainder of Lyeopene forms alpha-carotene and xenthophylls (3,3 '-dihydroxyl-alpha-carotene), another kind of oxy-compound.Carotenoid in the higher plant (being angiosperm) is found in plastid (being chloroplast(id) and chromoplast).Plastid is intracellular storage body, is different from the unitary film of vacuole, and plastid is wrapped up by duplicature.Plastid such as chloroplast(id) can also contain its oneself DNA and rrna, can duplicate and synthesize some its oneself protein independently.Therefore plastid has mitochondrial several characteristic.In leaf, carotenoid is present in the basal granule of chloroplast(id) usually, and here carotenoid provides optical protection function.β-Hu Luobusu and xenthophylls are main carotenoid, and epoxidised carotenoid zeaxanthin diepoxide and Neoxanthine are only to exist on a small quantity.Carotenoid accumulates in developmental petal chromoplast, and chlorophyll does not appear in developmental petal usually.As in petal, form chromoplast along with growing from chloroplast(id), carotenoid comes across in the fruit chromoplast.The enzyme that most participation phytoenes are converted into carotene and xenthophylls is variable embrane-associated protein, in case its activity is just lost in dissolving.In corn, carotenoid is present in horny endosperm (74%-86%), powdery endosperm (9%-23%), and in the plumule and wheat bran of corn grain.

[0007.0.10.10] at present only a few plant be widely used in the production of commercialization dyeing carotenoid.Yet, what the carotenoid synthetic productivity that dyes in most these plants was relatively low and costliness resulting carotenoid is produced.The dried mary bush petal and the mary bush petal enriched material that obtain from so-called xenthophylls mary bush are used as fodder additives so that increase the yellow of yolk and baking skin poultry industry.The tinting strength of mary bush petal meals depends on to a great extent and is known as the carotenoid component that xenthophylls is mainly lutein ester.The xenthophylls zeaxanthin that also is found in the mary bush petal has shown it is effective baking pigment, produces highly acceptable yellow to yellowish-orange.In xenthophylls, pigment xenthophylls and zeaxanthin content in the mixture that commerce can get is the abundantest.The structural formula of xenthophylls and zeaxanthin shows below.Carotenoid has been found that in the storage organ and petal of multiple higher plant.For example, the mary bush petal accumulates a large amount of esterified xanthophylls as its main xenthophylls carotenoid (about 75% to surpassing 90%), and has esterified zeaxanthin more on a small quantity.Except xenthophylls and zeaxanthin, the mary bush petal generally also presents the β-Hu Luobusu and the esterified xanthophylls of small amount of accumulation, but do not produce or accumulate canthaxanthin or astaxanthin, this is to form enzyme because lack 4-ketone-β-ionene ring in naturally occurring mary bush or its heterozygote.

A kind of method that [0008.0.10.10] improves biosynthetic throughput is to use recombinant DNA technology.Therefore, people expect to produce dyeing carotenoid, and utilize usually and determine the latest developments of carotenoid biosynthesizing from the β-Hu Luobusu to xenthophylls, so that the production of control carotenoid.The production of this type allows controlling quality, quantity and the suitableeest and the most effective selection of producing biology.The latter is even more important for commercially producing economics, so be useful to the human consumer.The method of recombinant DNA technology has been used some years, and these technology are used for improving the particularly production of xenthophylls in the algae or in the plant of microorganism by increase different xenthophylls biosynthesis genes and research to xenthophylls production.For example reported that 5 kinds of ketocarotinoids such as xenthophylls astaxanthin can result from the nectary of rotaring gene tobacco plant.Transform those transgenic plant of preparation by the tobacco plant that uses carrier to carry out the agrobacterium tumefaciens mediation, wherein carrier comprise from the ketolase encoding gene of H.pluvialis called after crtO and from the Pds gene of tomato as the promotor and the leader sequence of encoding.Those investigators claim that the Pds gene can instruct transcript and expression in the plant tissue that contains chloroplast(id) and/or chromoplast.Those results show that being found in the carotenoid that transforms about 75% in the plant flowers contains ketone group.In addition, in corn, identify crtB (Psy), octahydro lycopene desaturase (Pds) and-the carotene desaturase, and demonstrate the important control point that the PSY activity is a regulating flow.

Reported the gene that is suitable for microorganism and changes (U.S. Patent number 6,150,130WO99/61652).Two different genes that carotenoid β-ionene cyclic cpds can be converted into astaxanthin from the green alga Haematocoocus Pluvialls, separated obtain zeaxanthin or-carotene also is found in marine bacteria Agrobacterium aurantiacum, Alcaligenes PC-1, Erwiniauredovora.A.aurantiacum crtZ gene has been imported the intestinal bacteria transformant of accumulation content full cis-beta-carotene.So the transformant that forms produces zeaxanthin.The gene cluster that coding is used for the enzyme of carotenoid biosynthetic pathway also obtains from the red bacterial clone of purple photosynthesis bacterial capsule.Being used for carrying out the biosynthetic similar gene cluster of carotenoid from general precursor such as farnesyl pyrophosphate and geranyl tetra-sodium also obtains from non-photosynthetic bacterium Erwinia herbicola clone.And another kind of carotenoid biosynthesis gene bunch obtains from Erwinia uredovora clone.About be transformed into behind the higher plant cell by the enzyme of enzyme of other biological coding and A.aurantiacum external or intravital behavior whether similar remain the position with uncertain.

[0009.0.10.10] is except above-mentioned relevant carotenoid biology importance, for example in vision, osteogenesis, reproduction, immunologic function, genetic expression, embryo's expression, cell fission and cytodifferentiation, and breathe, should also be mentioned that in the world the popular scope of vitamin A deficiency from 100-250 1,000,000 children, and estimate have the 250.000-500.000 children to lose one's sight every year because of vitamin A deficiency.Therefore, will be favourable if can obtain to produce algae or other microorganism of a large amount of β-Hu Luobusus, β-cryptoxanthin, xenthophylls, zeaxanthin or other carotenoid.The biology that only produces xenthophylls on a small quantity or not may also be favourable, because enough for example one or more suitable '-hydroxylase genes of this type of bioenergy and/or suitable ketolase gene transform so that produce cryptoxanthin, zeaxanthin or astaxanthin.The present invention's discussion hereinafter relates to the protokaryon or the eukaryotic microorganisms of conversion like this in some embodiments.If it also is favourable can obtaining mary bush or other plant, a large amount of β-Hu Luobusus of the flower growth of these plants, β-cryptoxanthin, xenthophylls, zeaxanthin or other carotenoid.The biology that only produces xenthophylls on a small quantity or not may also be favourable because can be enough one or more suitable '-hydroxylase genes of this type of plant and/or suitable ketolase gene transform from the spending of for example resultant transformant generation cryptoxanthin, zeaxanthin or astaxanthin.The present invention's discussion hereinafter relates to plant transformed like this in some embodiments.

[0010.0.10.10] therefore, the quality that improves food and animal-feed is the vital task of food and fodder industry.This is inevitable, and is limited because for example be present in the xenthophylls as mentioned above of plant and certain micro-organisms for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is the spectrum of the carotenoid in the balanced diet as far as possible, though a large amount of excessive specific concentrations that surpass in the food of some carotenoid only have some positive effects.Further improve the quality unique possible be by adding other limited carotenoid.

[0011.0.10.10] therefore must add one or more carotenoid to be fit to biological balance mode for the quality of food and animal-feed.

[0012.0.10.10] therefore, still exist great demand new and more suitable gene, described genes encoding participates in carotenoid, for example xenthophylls such as β-cryptoxanthin or zeaxanthin or astaxanthin biological synthetic enzyme and can produce them and not form unnecessary byproduct in the particular industry scale.Selecting to be used for biosynthetic gene, above-mentioned two features are particularly importants.On the one hand, but still need to obtain the development of the highest intrinsic energy carotenoid such as xenthophylls, on the other hand, produce byproduct in process of production as few as possible.

[0013.0.0.10] sees [0013.0.0.0]

[0014.0.10.10] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is an xenthophylls.Therefore, in the present invention, term " fine chemicals " is meant " xenthophylls " as used herein.In addition, in another embodiment, term " fine chemicals " also refers to comprise the fine chemicals composition of xenthophylls as used herein.

An embodiment, term " xenthophylls " or " fine chemicals " or " each fine chemicals " meaning are meant at least a active chemical compound of the xenthophylls that is selected from zeaxanthin or cryptoxanthin that has [0015.0.10.10].

In one embodiment, term " fine chemicals " meaning is meant xenthophylls.In one embodiment, depend on the employed environment of term, term " fine chemicals " meaning is meant β-cryptoxanthin or zeaxanthin.In whole specification sheets, term " fine chemicals " meaning is meant xenthophylls, particularly β cryptoxanthin or zeaxanthin, its salt, ester, thioesters or free form or be bonded to other compound such as the form of sugar or glycopolymers (as glucosides, for example bioside).In one embodiment, term " fine chemicals " meaning is meant β-cryptoxanthin and zeaxanthin or its salt or its ester of free form or is bonded to the form of glucosides (for example bioside).In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning is meant at least a active chemical compound of above-mentioned fine chemicals that has.

[0016.0.10.10] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0050, b0851, b2211, b3926, b0986, b3684, b4401 and/or YCL040W protein, perhaps b2699 and/or YHR055C protein perhaps have the protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 3rd row, 103-108 or 468-471 are capable; With

(b) in allowing described biology, produce fine chemicals be β-cryptoxanthin or zeaxanthin comprise β-cryptoxanthin or the condition of the fine chemicals of zeaxanthin under make biological growth.

Therefore, the present invention relates to a kind of method, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, be respectively 103-108 or 468-471 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, be respectively 103-108 or 468-471 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce fine chemicals be β-cryptoxanthin or zeaxanthin comprise β-cryptoxanthin or the condition of the fine chemicals of zeaxanthin under make biological growth.

[0016.1.0.10] therefore, term " fine chemicals " refers to " β-cryptoxanthin " or its homologue relevant with Table I-IV 103-106 and/or the capable listed full sequence of 468-471, and " zeaxanthin " or its homologue relevant with the listed full sequence of 108 every trades with Table I-IV the 107th.

Therefore, term " fine chemicals " can refer to " zeaxanthin " or " cryptoxanthin " according to environment and context.For being meant, the meaning of illustrating term " each fine chemicals " " cryptoxanthin " and/or " zeaxanthin " from the listed sequence of context also can use term " each fine chemicals ".

Term " β-cryptoxanthin " and " cryptoxanthin " use as equivalent terms.

[0017.0.0.10] to [0018.0.0.10]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.10.10] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 103-108 or 468-471 are capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 103-108 or 468-471 were capable.

[0020.0.10.10] is surprisingly found out that, β-cryptoxanthin (with regard to described protein and homologue thereof and coding nucleic acid molecule (particularly as shown in Table I or II the 3rd row, 103-106 and/or 468-471 are capable), being " fine chemicals " or " each fine chemicals ") content that at least a protein is given the plant that transforms at transgene expression in the Arabidopis thaliana among yeast saccharomyces cerevisiae protein YCL040W and/or e. coli k12 protein b0050, b0851, b2211, b3926, b0986, b3684 and/or the b4401 increases.

Be surprisingly found out that, zeaxanthin (with regard to described protein and homologue thereof and coding nucleic acid molecule (particularly shown in Table I or II the 3rd row, the 107th and 108 row), being " fine chemicals " or " each fine chemicals ") content that yeast saccharomyces cerevisiae protein YHR055C and/or e. coli k12 protein b2699 give the plant that transforms at transgene expression in the Arabidopis thaliana increases.

[0021.0.0.10] sees [0021.0.0.0]

The sequence of [0022.0.10.10] e. coli k12 b0050 has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the conservative protein matter of potential participation apaG protein superfamily protein-protein interaction.Therefore, in one embodiment, the inventive method comprises as shown here having a proteolytic degradation from colibacillary, protein modification, by ubiquitinization, remove the active protein of ubiquitin modification and/or protein bound, the conservative protein matter of particularly potential participation protein-protein interaction, the conservative protein matter of particularly potential participation apaG protein superfamily protein-protein interaction or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be xenthophylls, particularly increase β-cryptoxanthin, the quantity of preferred free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, the activity of the conservative protein matter of potential participation apaG protein superfamily protein-protein interaction is enhanced or produces, and for example is enhanced or produces from the conservative protein matter of colibacillary potential participation apaG protein superfamily protein-protein interaction or the activity of its homologue.

The sequence of e. coli k12 b0851 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the oxygen insensitivity NADPH nitroreductase of NADPH-flavine oxydo-reductase homologue superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having a nitrogen metabolism from colibacillary, sulfo-is thanked, electron transport and film correlation energy are preserved, coerce reaction, electronics/hydrogen vehicle, C-compound and carbohydrate utilization and/or VITAMIN, cofactor and prothetic group utilize active protein and/or as the protein of resistance protein, oxygen insensitivity NADPH nitroreductase particularly, particularly the oxygen insensitivity NADPH nitroreductase of NADPH-flavine oxydo-reductase homologue superfamily or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be xenthophylls, particularly increase β-cryptoxanthin, the quantity of preferred free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, the activity of the oxygen insensitivity NADPH nitroreductase of NADPH-flavine oxydo-reductase homologue superfamily is enhanced or produces, and for example is enhanced or produces from the oxygen insensitivity NADPH nitroreductase of colibacillary NADPH-flavine oxydo-reductase homologue superfamily or the activity of its homologue.

The sequence of e. coli k12 b2211 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the ATP-binding transport albumen of not determining the ATP-binding cassette superfamily of ATP-binding cassette protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having a Nucleotide combination from colibacillary, the active protein of abc transport body and/or cell traffic and transporting mechanism, the ATP-binding transport albumen of ATP-binding cassette superfamily particularly, particularly do not determine the ATP-binding transport albumen of ATP-binding cassette superfamily of ATP-binding cassette protein superfamily or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be xenthophylls, particularly increase β-cryptoxanthin, the quantity of preferred free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, the proteic activity of ATP-binding transport of determining the ATP-binding cassette superfamily of ATP-binding cassette protein superfamily is enhanced or produces, and for example is enhanced or produces from the colibacillary not ATP-binding transport albumen of the ATP-binding cassette superfamily of definite ATP-binding cassette protein superfamily or the activity of its homologue.

The sequence of e. coli k12 b3926 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the glycerol kinase of xylulokinase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary C-of having compound and sugar metabolism, the phosphatide biosynthesizing, the biosynthesizing of glycolipid class, unspecified signal transduction, the active protein of enzyme Mediated Signal Transduction and/or phosphotransferase system, glycerol kinase particularly, the purposes of the glycerol kinase of xylulokinase superfamily or its homologue particularly, it is used for producing each fine chemicals in biological or its part as described, be xenthophylls, particularly increase β-cryptoxanthin, the quantity of preferred free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, the activity of the glycerol kinase of xylulokinase superfamily is enhanced or produces, and for example is enhanced or produces from the glycerol kinase of colibacillary xylulokinase superfamily or the activity of its homologue.

The sequence of e. coli k12 b0986 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the lipoprotein of putative protein matter b1706 superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary lipoprotein, the particularly lipoprotein of putative protein matter b1706 superfamily or the purposes of its homologue, it is used for producing each fine chemicals, being xenthophylls in biological or its part as described, particularly increases β-cryptoxanthin, the preferred quantity of free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, lipoprotein, the particularly activity of the lipoprotein of putative protein matter b1706 superfamily are enhanced or produce, and for example are enhanced or produce from the lipoprotein of colibacillary lipoprotein, particularly putative protein matter b1706 superfamily or the activity of its homologue.

The sequence of e. coli k12 b3684 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be GntR family transcriptional.Therefore, in one embodiment, the inventive method comprises that as shown here to have the control of transcribing active from colibacillary, C-compound and carbohydrate utilization are regulated active, dna binding activity, the transcription repressor activity, lipid, lipid acid and/or isoprenoid metabolism are regulated activity and/or C-compound and carbohydrate utilization and are regulated active protein, transcriptional for example, the purposes of GntR family transcriptional or its homologue particularly, it is used for producing each fine chemicals in biological or its part as described, be xenthophylls, particularly increase β-cryptoxanthin, the quantity of preferred free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, described activity for example transcriptional, the particularly activity of GntR family transcriptional is enhanced or produces, and for example the activity from colibacillary transcriptional, particularly GntR family transcriptional or its homologue is enhanced or produces.

The sequence of e. coli k12 b4401 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be that regulatory gene is replied by OmpR family.Therefore, in one embodiment, the inventive method comprises that as shown here to have the control of transcribing active from colibacillary, unspecified signal transduction activity, the two-pack signal transduction system is as replying the activity of regulatory gene composition, transcriptional activation agent activity, the respiratory regulation activity, the active protein of aerobic respiration activity and/or anaerobic respiration, for example reply regulatory gene, particularly the purposes of regulatory gene or its homologue is replied by OmpR family, it is used for producing each fine chemicals in biological or its part as described, be xenthophylls, particularly increase β-cryptoxanthin, the quantity of preferred free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, described activity is for example replied regulatory gene, particularly OmpR family and is replied the activity of regulatory gene and be enhanced or produce, and for example replys the activity of regulatory gene or its homologue and is enhanced or produces from colibacillary regulatory gene, particularly the OmpR family replied.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the DNA chain exchange with proteolytic enzyme and nuclease and the recombinant protein of recombinant protein recA superfamily.Therefore, in one embodiment, the inventive method comprises as shown here recombinating and the DNA repairing activity from the colibacillary DNA of having, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, the purposes of recombinant protein recA superfamily or its homologue particularly, it is used for producing each fine chemicals in biological or its part as described, be xenthophylls, particularly increase zeaxanthin, the quantity of preferred free or combining form zeaxanthin.In one embodiment, in the methods of the invention, described activity proteolytic enzyme and nuclease for example, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, particularly the activity of recA superfamily recombinant protein is enhanced or produces, and for example the activity from colibacillary these protein or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YCL040W has been published in Oliver, S.G., etc., Nature 357 (6373), 38-46 (1992) and Goffeau etc., and Science 274 (5287), 546-547,1996, and its activity to be defined as be the glucokinase of hexokinase superfamily.Therefore, in one embodiment, the inventive method comprises that have C-compound and carbohydrate utilization activity, C-compound or carbohydrate transport activity, glycolysis-activity and/or glyconeogenesis activity and/or cell as shown here import active protein, hexokinase particularly, the purposes of preferred glucokinase or its homologue, it is used for producing each fine chemicals, being xenthophylls in biological or its part as described, particularly increases the quantity of free or combining form β-cryptoxanthin.In one embodiment, in the methods of the invention, the described activity for example activity of glucokinase is enhanced or produces, and for example is enhanced or produces from the glucokinase of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YHR055C has been published in Johnston, M etc., Science 265 (5181), 2077-2082 (1994) and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be the copper bond sulfoprotein of metallothionein(MT) superfamily.Therefore, in one embodiment, the inventive method comprises the protein with detoxicating activity as shown here, the purposes of metallothionein(MT), preferably copper bond sulfoprotein or its homologue particularly, it is used for producing each fine chemicals, being xenthophylls in biological or its part as described, particularly increases the quantity of free or combining form zeaxanthin.In one embodiment, in the methods of the invention, the described activity for example activity of metallothionein(MT), preferably copper bond sulfoprotein is enhanced or produces, and for example the activity from metallothionein(MT), preferably copper bond sulfoprotein or its homologue of yeast saccharomyces cerevisiae is enhanced or produces.

The homologous compound (=homologue) of [0023.0.10.10] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with protein shown at least a Table II the 3rd row, 103-106 and/or 468-471 are capable, for example have by contain Table I the 5th or 7 row, 103-106 and/or 468-471 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in Table II the 3rd row, the 103rd row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred β-cryptoxanthin content increases.In one embodiment, homologue is the homologue that has sequence shown in Table I or II the 7th row, the 103rd row respectively.In one embodiment, shown in Table II the 3rd row, the 103rd row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 103rd row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 103rd row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 103rd row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 103rd row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 103rd row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 103rd row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, 104-106 or 468-471 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred cryptoxanthin content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 104-106 or 468-471 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, 104-106 or 468-471 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, 104-106 or 468-471 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, 104-106 or 468-471 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, 104-106 or 468-471 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, 104-106 or 468-471 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, 104-106 or 468-471 capable shown in the homologue of polypeptide be to have same or similar activity (particularly giving β in biology or its part-cryptoxanthin content increases) and from the homologue of Escherichia.

In one embodiment, shown in Table II the 3rd row, the 107th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred zeaxanthin content increase.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 107th row.In one embodiment, shown in Table II the 3rd row, the 107th row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 107th row is from Ascomycota.In one embodiment, Table II the 3rd row, the 107th row 65-67 capable shown in the homologue of polypeptide from yeast.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 107th row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 107th row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 107th row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 107th row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II the 3rd row, the 108th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred zeaxanthin content increase.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 108th row.In one embodiment, shown in Table II the 3rd row, the 108th row in the polypeptide homologue of any one from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 108th row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 108th row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 108th row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 108th row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 108th row is to have same or similar activity (particularly active improve give zeaxanthin content increases in biology or its part) and from the homologue of Escherichia.

[0023.1.0.10] as the homologue of the polypeptide shown in Table II the 3rd row, 103-108 or 468-471 are capable can be by Table I the 7th row, 103-108 or 468-471 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 103-108 or 468-471 capable shown in polypeptide.As the homologue of polypeptide shown in Table II the 3rd row, 103-108 or 468-471 are capable can be by Table I the 7th row, 103-108 or 468-471 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 103-108 or 468-471 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, 103-106 and/or 468-471 are capable can be have increase cryptoxanthin content and/or quantity is active, by Table I the 7th row, be respectively 103-106 and/or 468-471 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 103-106 and/or 468-471 capable shown in polypeptide.The homologue of polypeptide shown in Table II the 3rd row, 103-106 and/or 468-471 are capable can be have increase cryptoxanthin content and/or quantity is active, by Table I the 7th row, 103-106 and/or 468-471 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 103-106 and/or 468-471 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, the 107th and/or 108 row can be have increase zeaxanthin content and/or quantity is active, by polypeptide shown in Table I the 7th row, the polypeptide that is respectively nucleic acid molecule encoding shown in the 107th and/or 108 row or Table II the 7th row, the 107th and/or 108 row.The homologue of polypeptide shown in Table II the 3rd row, the 107th and/or 108 row can be have increase zeaxanthin content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, the 107th and/or 108 row or Table II the 7th row, the 107th and/or 108 row.

[0024.0.0.10] sees [0024.0.0.0]

[0025.0.10.10] is according to the present invention, lutein levels increases in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, particularly β-cryptoxanthin level or zeaxanthin level increase respectively, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II the 3rd row, be respectively 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, be respectively 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in any one proteinic biology or the enzyme activity, if promptly with Table II the 3rd row, any one yeast saccharomyces cerevisiae protein shown in the 103rd row or the 107th row is compared and/or is listed as with Table II the 3rd, 104-106 capable or the 108th the row and/or 468-4716 capable shown in any one e. coli k12 protein compare, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as each fine chemicals of increase in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

[0025.1.0.10] sees [0025.1.0.0]

[0025.2.0.10] sees [0025.2.0.0]

[0026.0.0.10] to [0033.0.0.10]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.10.10] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein or by Table I the 5th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the protein of nucleic acid molecule encoding or its homologue (as Table I the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in homologue) active protein expression level or active aspect difference, and its difference aspect biological chemistry or genetics reason.Therefore it shows each fine chemicals quantity that increases.

[0035.0.0.10] to [0044.0.0.10]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.10.10] in one embodiment, at e. coli k12 protein b0050 or its homologue, for example potential participation protein-protein interaction conservative protein matter, under the situation that the activity of particularly potential participation apaG protein superfamily protein-protein interaction conservative protein matter (for example shown in Table I the 5th or 7 row, the 468th row) is enhanced, preferably, give each fine chemicals, preferred β-cryptoxanthin be increased in 42% and 84% or more between.

In one embodiment, at e. coli k12 protein b0851 or its homologue, oxygen insensitivity NADPH nitroreductase for example, particularly under the situation that the activity of the oxygen insensitivity NADPH nitroreductase of NADPH-flavine oxydo-reductase homologue superfamily (shown in Table I the 5th for example or 7 row, the 469th row) is enhanced, preferably, give each fine chemicals, preferred β-cryptoxanthin be increased in 57% and 76% or more between.

In one embodiment, at e. coli k12 protein b2211 or its homologue, ATP-binding cassette superfamily ATP-binding transport albumen for example, particularly do not determine under the situation that the activity of the ATP-binding cassette superfamily ATP-binding transport albumen (for example shown in Table I the 5th or 7 row, the 470th row) of ATP-binding cassette protein superfamily is enhanced, preferably, give each fine chemicals, preferred β-cryptoxanthin be increased in 5% and 26% or more between.

In one embodiment, at e. coli k12 protein b3926 or its homologue, glycerol kinase for example, under the situation that the activity of particularly super xylulokinase family glycerol kinase (for example shown in Table I the 5th or 7 row, the 471st row) is enhanced, preferably, give each fine chemicals, preferred β-cryptoxanthin increases about 33% or higher.

In one embodiment, at e. coli k12 protein b0986 or its homologue, lipoprotein for example, particularly under the activity situation about being enhanced of the lipoprotein of putative protein matter b1706 superfamily (for example shown in Table I the 5th or 7 row, the 104th row), preferably, give each fine chemicals, preferred β-cryptoxanthin be increased in 33% and 40% or more between.

Therefore, in one embodiment, be enhanced in the activity of e. coli k12 protein b3684 or its homologue.Therefore, in one embodiment, have that the control of transcribing is active, C-compound and carbohydrate utilization regulate activity, dna binding activity, transcription repressor activity, lipid, lipid acid and/or isoprenoid metabolism adjusting activity and/or C-compound and carbohydrate utilization and regulate active protein, transcriptional for example, particularly under the activity situation about being enhanced of GntR family transcriptional (for example shown in Table I the 5th or 7 row, the 105th row), preferably, give each fine chemicals, preferred β-cryptoxanthin be increased in 35% and 67% or more between.

In one embodiment, at e. coli k12 protein b4401 or its homologue, it is active for example to have the control of transcribing, unspecified signal transduction activity, the two-pack signal transduction system is for example replied regulatory gene composition activity, transcriptional activation agent activity, the respiratory regulation activity, the active protein of aerobic respiration activity and/or anaerobic respiration, for example reply regulatory gene, particularly regulatory gene is replied (for example as Table I the 5th or 7 row by OmpR family, shown in the 106th row) activity situation about being enhanced under, preferably, give each fine chemicals, preferred β-cryptoxanthin be increased in 48% and 60% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YCL040W or its homologue, for example have C-compound and carbohydrate utilization activity, C-compound or carbohydrate transport activity, glycolysis-activity and/or glyconeogenesis activity and/or cell and import active protein, hexokinase particularly, under the situation that the activity of preferred glucokinase (for example shown in Table I the 5th or 7 row, the 103rd row) is enhanced, preferably, give each fine chemicals, preferred β-cryptoxanthin be increased in 36% and 51% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example DNA recombinates and the DNA repairing activity, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, particularly recombinant protein recA superfamily is (for example as Table I the 5th or 7 row, shown in the 108th row) activity situation about being enhanced under, preferably, give each fine chemicals, preferred zeaxanthin be increased in 25% and 48% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YHR055C or its homologue, the protein that for example has detoxicating activity, metallothionein(MT) particularly, under the activity situation about being enhanced of preferably copper bond sulfoprotein or its homologue (for example shown in Table I the 5th or 7 row, the 107th row), preferably, give each fine chemicals, preferred zeaxanthin be increased in 05% and 38% or more between.

[0046.0.10.10] in one embodiment, at e. coli k12 protein b0050 or its homologue, for example potential participation protein-protein interaction conservative protein matter, under the situation that the activity of particularly potential participation apaG protein superfamily protein-protein interaction conservative protein matter (for example shown in Table I the 5th or 7 row, the 468th row) is enhanced, preferably, give each fine chemicals and other carotenoid, preferred xenthophylls increase.

In one embodiment, at e. coli k12 protein b0851 or its homologue, oxygen insensitivity NADPH nitroreductase for example, particularly under the situation that the activity of the oxygen insensitivity NADPH nitroreductase of NADPH-flavine oxydo-reductase homologue superfamily (shown in Table I the 5th for example or 7 row, the 469th row) is enhanced, preferably, give each fine chemicals and other carotenoid, preferred xenthophylls increase.

In one embodiment, at e. coli k12 protein b2211 or its homologue, the ATP-binding transport albumen of ATP-binding cassette superfamily for example, particularly do not determine under the situation that the activity of the ATP-binding transport albumen (for example shown in Table I the 5th or 7 row, the 470th row) of the ATP-binding cassette superfamily of ATP-binding cassette protein superfamily is enhanced, preferably, give each fine chemicals and other carotenoid, preferred xenthophylls increase.

In one embodiment, at e. coli k12 protein b3926 or its homologue, glycerol kinase for example, particularly under the activity situation about being enhanced of xylulokinase superfamily glycerol kinase (for example shown in Table I the 5th or 7 row, the 471st row), preferably, give each fine chemicals and other carotenoid, preferred xenthophylls increase.

In one embodiment, at e. coli k12 protein b0986 or its homologue, for example under the activity situation about being enhanced of lipoprotein, the particularly lipoprotein of putative protein matter b1706 superfamily (for example shown in Table I the 5th or 7 row, the 104th row); Preferably, give each fine chemicals and other carotenoid, preferred xenthophylls increase.

In one embodiment, at e. coli k12 protein b3684 or its homologue, for example under the situation that the activity of GntR family transcriptional is enhanced; Preferably give each fine chemicals and other carotenoid, preferred xenthophylls increase.Therefore, in one embodiment, the inventive method comprises that as shown here having transcribes that control is active, C-compound and carbohydrate utilization are regulated activity, dna binding activity, transcription repressor activity, lipid, lipid acid and/or isoprenoid metabolism adjusting activity and/or C-compound and carbohydrate utilization and regulated active protein, transcriptional for example, the purposes of GntR family transcriptional (for example shown in Table I the 5th or 7 row, the 105th row) particularly, it preferably gives each fine chemicals and other carotenoid, preferred xenthophylls increases.

In one embodiment, at e. coli k12 protein b4401 or its homologue, it is active for example to have the control of transcribing, unspecified signal transduction activity, the two-pack signal transduction system is for example replied regulatory gene composition activity, transcriptional activation agent activity, the respiratory regulation activity, the active protein of aerobic respiration activity and/or anaerobic respiration, for example reply regulatory gene, particularly regulatory gene is replied (for example as Table I the 5th or 7 row by OmpR family, shown in the 106th row) activity situation about being enhanced under, preferably give each fine chemicals and other carotenoid, preferred xenthophylls increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YCL040W or its homologue, for example have C-compound and carbohydrate utilization activity, C-compound or carbohydrate transport activity, glycolysis-activity and/or glyconeogenesis activity and/or cell and import active protein, hexokinase particularly, under the situation that the activity of preferred glucokinase (for example shown in Table I the 5th or 7 row, the 103rd row) is enhanced, preferably give each fine chemicals and other carotenoid, preferred xenthophylls increases.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example DNA recombinates and the DNA repairing activity, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the protein that particularly has proteolytic enzyme and nuclease, particularly exchange of recombinant protein recA superfamily DNA chain and recombinant protein are (for example as Table I the 5th or 7 row, shown in the 108th row) activity situation about being enhanced under, preferably give each fine chemicals and other carotenoid, preferred xenthophylls increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YHR055C or its homologue, the protein that for example has detoxicating activity, metallothionein(MT) particularly, under the activity situation about being enhanced of preferably copper bond sulfoprotein or its homologue (for example shown in Table I the 5th or 7 row, the 107th row), preferably give each fine chemicals and other carotenoid, preferred xenthophylls increases.

[0047.0.0.10] to [0048.0.0.10]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.10.10] has to give and improves each fine chemicals cryptoxanthin quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in 103-107 and/or 468-471 are capable or as Table II the 5th or 7 row, its function homologue shown in 103-106 and/or 468-471 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 103-106 and/or 468-471 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that β-cryptoxanthin level increases of giving described in the literary composition.

Have to give and improve zeaxanthin quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the sequence of consensus sequence shown in Table IV the 7th row, the 107th and 108 row and/or shown in Table II the 5th or 7 row, the 107th or 108 row its function homologue described in polypeptide or the literary composition or by the sequence of the coded polypeptide of nucleic acid molecule that this paper characterized or nucleic acid molecule of the present invention (for example by nucleic acid molecule or its function homologue as herein described shown in Table I the 5th or 7 row, the 107th or 108 row), and have the activity that the zeaxanthin level of giving described in the literary composition increases.

[0050.0.10.10] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, the sylvite of cryptoxanthin or zeaxanthin or sodium salt, perhaps their ester or glucoside (for example diglucoside).

[0051.0.10.10] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), the composition that for example comprises xenthophylls, particularly cryptoxanthin or zeaxanthin.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of multiple xenthophylls, particularly β-cryptoxanthin or zeaxanthin.

[0052.0.0.10] sees [0052.0.0.0]

[0053.0.10.10] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein give the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein or its homologue (for example Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in) active polypeptide) express and increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example have as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein or its homologue (for example Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein is given has the active of each fine chemicals of the raising described in the literary composition, (for example have by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide as Table II the 3rd row, protein or its homologue shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 (for example Table II the 5th or 7 row, shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471) active polypeptide) express to improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by nucleic acid molecule of the present invention or the coded protein of the inventive method nucleic acid molecule used therefor, polypeptide perhaps of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, protein or its homologue shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 (for example Table II the 5th or 7 row, shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471) active polypeptide) express to improve;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein or its homologue (for example Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in) active polypeptide) express and improve;

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein or its homologue (for example Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, and described nucleic acid molecule encoding has the active of each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method as Table II the 3rd row, protein or its homologue shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 (for example Table II the 5th or 7 row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in) active polypeptide);

(h) by add positive Expression element or remove negative Expression element improve code book invention polypeptide or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein or its homologue (for example Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.10.10] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having as Table II the 5th or 7 row, be respectively protein or its homologue shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 (for example Table II the 5th or 7 row, be respectively polypeptide shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471) give each fine chemicals after active expression of polypeptides or the activity and increase.

[0055.0.0.10] to [0067.0.0.10]: see that [0055.0.0.0] is to [0067.0.0.0]

[0068.0.10.10] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of xenthophylls, particularly β-cryptoxanthin or zeaxanthin.

[0069.0.10.10] sees [0069.0.0.0]

[0070.0.10.10] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used expression of polypeptides of the inventive method, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example favourable carotenoid, particularly the xenthophylls composition for example contains high level (from the physiology of nutrition angle) carotenoid, the composition of xenthophylls such as β-cryptoxanthin or zeaxanthin particularly.

[0071.0.0.10] sees [0071.0.0.0]

[0072.0.10.10] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has other carotenoid except β-cryptoxanthin or zeaxanthin, for example carotene or xenthophylls, particularly for example xenthophylls, Lyeopene, alpha-carotene or β-Hu Luobusu or its precursor compound are the compound of β-cryptoxanthin or zeaxanthin, particularly astaxanthin for ketocarotinoids, hydrogen carotenoid.

[0073.0.10.10] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or the used polypeptide of the inventive method or its homologue (for example Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and optional other is free and/or bonded carotenoid, particularly xenthophylls astaxanthin for example.

[0074.0.10.10] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded or free and each fine chemicals of bonded, if and dissociate or/and bonded carotenoid, particularly xenthophylls astaxanthin for example as selecting to produce, reclaim and expect to separate other.

[0075.0.0.10] to [0077.0.0.10]: see that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.10.10] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes, for example according to US 6,380,442; US 6,329,557, and US 6,329,432, US6, and 316,012, US 6,309, and 883; US 6,291,533, and US 6,291,294, and US 6,287,615, and US 6,262,284, US 6,262,284, and US 6,261,622, and US 6,261,598, US6,235,315, US 6,224,876, and US 6,221, and 417, US 6,221,412, US6, and 218,436, US 6,207,409, and US 6,319,872, US 6,132,790, and US 6,124, and 113US 6,110,478, US 6,093,348, and US 6,087,152 or US 6,056,962 in disclose.The pharmaceutical composition or other composition that comprise zeaxanthin are described in the following patent: US6 for example, 383,523, US 6,368, and 621, US 6,362, and 221, US 6,348, and 200, US 6,316,012:Cosmetic or pharmaceutical composition comprising, in combination, aperoxidase and an anti-singlet oxygen agent; 6,296,880:Pharmaceuticalcompositions and methods for managing skin conditions; US 6,296,877:Stable, aqueous dispersions and stable, water-dispersible dry xanthophyllpowder, their production and use; US 6,261,598:Carotenoid formulations, comprising a mixture of B-carotens, lycopene and lutein; US 6,248,378:Enhanced food products; US 6,248,374:Stabilized food additive.Separation method is described in following patent, and for example US 6,380,442, and US 6,362,221:Compositionscontaining natural lycopene and natural tocopherol; US 6,291,204:Fermentative carotenoid production; US 6,262,284:Process for extractionand purification of lutein, zeaxanthin and rare carotenoids from marigoldflowers and plants; Perhaps US 6,224,876:Isolation and formulations ofnutrient-rich carotenoids.The document description of being quoted some embodiment preferred.Some nonrestrictive advantageous embodiment have been described in described application.Can as described in the above-mentioned application or as other method well known to those skilled in the art, for example at Methods in Enzymology:Carotenoids, Part A:Chemistry, Separation, Quantitation andAntioxidation, by John N Abelson or Part B, Metabolism, Genetics, described in the andBiochemistry or described hereinafter method come purified fermentation broth, tunning, plant or plant product.For these different methods of setting up gradually, product is the composition that contains xenthophylls, particularly zeaxanthin or β-cryptoxanthin or xenthophylls, particularly zeaxanthin or cryptoxanthin, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.10] to [0084.0.0.10]: see that [0079.0.0.0] is to [0084.0.0.0]

[0084.1.0.10] the present invention has also considered such embodiment, and promptly in each fine chemicals that produces, β-Hu Luobusu or other carotenoid precursors compound are present in the spending of selected flowering plant (for example, mary bush) as the host.The present invention has also considered such embodiment, and promptly the flower of host plant lacks β-Hu Luobusu or other carotenoid precursors, for example Vinca.In the one type plant of back, the DNA that is inserted (if lacking other) comprises the gene of coding carotenoid precursors (can biologically be transformed into the compound of β-Hu Luobusu) and ketolase and hydroxylase.

In one embodiment, preferred flowering plant includes but not limited to: Amaryllidaceae (Amaryllidaceae) (allium (Allium), Narcissus (Narcissus)); Apocynaceae (Apocynaceae) (Vinca (Catharanthus)); Composite family (Compositae or Compositae) (Aster (Aster), calendulin, China aster belong to (Callistephus), Cichorium, golden pheasant Chrysanthemum (Coreopsis), dahlia (Dahlia), Chrysanthemum (Dendranthema), variegated Chrysanthemum (Gazania), African daisy genus (Gerbera), Helianthus, Helichrysum (Helichrysum), Lactuca, Rudbeckia (Rudbeckia), Tagetes, youth-and-old-age genus (Zinnia)); Balsaminaceae (impatiens (Impatiens)); Begoniaceae (Begoniaceae) (begonia (Begonia)); Caryophyllaceae (Carnation (Dianthus)); Chenopodiaceae (Beta, spinach belong to (Spinacia)); Curcurbitaceae (Citrullus (Citrullus), Curcurbita genus, Cucumis (Cucumis)); Cruciferae (Alyssum (Alyssum), Btassica, Erysimum (Erysimum), Matthiola incana (Matthiola), Rhaphanus (Raphanus)); Gentianaceae (Gentinaceae) (Eustoma genus); Mang ox seedling section (Pelargonium); Gramineae (Graminae or Poaceae) (Avena, Hordeum (Horedum), Oryza, Panicum, Pennisetum (Pennisetum), annual bluegrass belong to (Poa), saccharum (Saccharum), Secale, jowar genus, Triticum, Zea); Euphorbiaceae (Euphorbia (poinsettia)); Labiatae (Labiatae) (Salvia (Salvia)); Pulse family (Glycine, Lathyrus (Lathyrus), Medicago (Medicago), Phaseolus, Pisum); Liliaceae (lilium (Lilium)); Lobeliaceae section (lobelia (Lobelia)); Malvaceae (Abelmoschus (Abelmoschus), Gossypium, mallow (Malva)); Plumbaginaceae (Plumbaginaceae) (Statice (Limonium)); Hua Shallot section (Polemoniaceae) (perennial pholx belongs to (Phlox)); Primulaceae (Primulaceae) (Cyclamen (Cyclamen)); Ranunculaceae (Aconitum (Aconitum), Anemone (Anemone), aquilegia (Aquilegia), kincup belong to (Caltha), staggerweed (Delphinium), Ranunculus (Ranunculus)); The Rosaceae (rose (Rosa)); Rubiaceae (Pentas genus); Scrophulariaceae (Scrophulariaceae) (fragrant painted bunting belongs to (Angelonia), antirrhinum (Antirrhinum), HUDIECAO belongs to (Torenia)); Solanaceae (Capsicum, tomato genus, Nicotiana, green winter Solanum (Petunia), Solanum); Umbelliferae (apium, Daucus (Daucus), Pastinaca belong to); Verbenaceae (Verbenaceae) (Verbena (Verbena), Lantana (Lantana)); Violaceae (Viola (Viola)).

[0085.0.10.10] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, be respectively 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, be respectively 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.10] to [0087.0.0.10]: see that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.10.10] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Because the nutritive value that for example is used for the plant of raise poultry depends on above-mentioned xenthophylls and as the total number of the xenthophylls in fodder energy source, so this is very important for the plant breeder.In addition, because the antioxidant level of plant milk extract depends on above-mentioned xenthophylls quantity and/or carotenoid quantity as antioxidant, so this production for make-up composition also is important.

[0088.1.0.10] sees [0088.1.0.0]

[0089.0.0.10] to [0090.0.0.10]: see that [0089.0.0.0] is to [0090.0.0.0]

[0091.0.10.10] therefore, the plant component foreign matter content is low as far as possible, and preferably other foreign matter contents are also low as far as possible, and the aforementioned xenthophylls, the particularly fine chemicals that obtain are pure form as far as possible.In these are used, the content of plant component favourable for by weight less than 10%, preferred 1%, more preferably 0.1%, very particularly preferably 0.01% or lower.

[0092.0.0.10] to [0094.0.0.10]: see that [0092.0.0.0] is to [0094.0.0.0]

[0095.0.10.10] advantageously, the pond that increases carotenoid, particularly xenthophylls, preferred cryptoxanthin or zeaxanthin by method of the present invention in genetically modified organism is to separate a large amount of each pure basically fine chemicals.

[0096.0.10.10] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals such as zeaxanthin or cryptoxanthin in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.Existing report, the inhibition that Lyeopene (Lyopene) is produced has increased the quantity of other xenthophylls in the cell.In addition, use zeaxanthin or cryptoxanthin can also increase the quantity of chemical described in the cell as the substrate inhibitory enzyme.For example, in one embodiment, if wish to produce the cryptoxanthin or the zeaxanthin of high quantity, the generation that then suppresses astaxanthin is favourable.

Other modification of the glucosides of [0097.0.10.10] zeaxanthin and β-cryptoxanthin, particularly bioside and zeaxanthin and cryptoxanthin is well known by persons skilled in the art.The content that increases each fine chemicals of bonded (for example to the modification of zeaxanthin and cryptoxanthin (particularly its glucosides, as bioside)) also is favourable.

[0098.0.10.10] in preferred embodiments, each fine chemicals be produce according to the present invention and carry out isolating where necessary.The method according to this invention produces other carotenoid, for example carotene or xenthophylls, particularly ketocarotinoids or hydrogen carotenoid, for example xenthophylls, Lyeopene, alpha-carotene or β-Hu Luobusu, perhaps each fine chemicals is the compound (for example astaxanthin) of its biosynthesizing precursor compound, perhaps its mixture or with the mixture of other carotenoid (particularly xenthophylls) be favourable.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.10.10].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition that comprises each fine chemicals.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.10.10] comprises the method according to this invention synthetic and comprises carotenoid, the transgenic plant of xenthophylls such as cryptoxanthin or zeaxanthin (perhaps astaxanthin (because it is synthetic from cryptoxanthin or zeaxanthin)) can advantageously directly be put on market as described, and do not need institute's synthetic carotenoid is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet, each fine chemicals that the method according to this invention produces can also with the form of its oils, fat, lipid as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free lutein from biology, advantageously from plant, separate.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.For example, contain xenthophylls oils, fat and/or lipid can need not heat cold be beaten or cold-press process obtains by being called.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because carotenoid is positioned at cell in the microorganism, must relate to separation to their recovery to biomass.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.Yet it is favourable avoiding this processing, because should processing can cause cell to lose most of carotenoid.

[0101.0.10.10] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications 0fHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.10.10] for example, xenthophylls, particularly β-cryptoxanthin or zeaxanthin can advantageously detect by HPLC, LC or GC separation method.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect the existence of the product that contains xenthophylls, particularly β-cryptoxanthin or zeaxanthin to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

[0103.0.10.10] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule have as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use have as Table III the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0103.1.10.10] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in polypeptide of sequence.

[0103.2.10.10.] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in polypeptide of sequence.

[0104.0.10.10] in one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in polypeptide of sequence.

[0105.0.0.10] to [0107.0.0.10]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.10.10] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, from Table II the 5th or 7 row, aminoacid sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 is derived or from containing Table IV the 7th row, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, perhaps its coding has as Table II the 3rd, 5 or 7 row, the enzymic activity of polypeptide or bioactive polypeptide shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 or for example to give each fine chemicals behind its expression or active the increasing be derivative or the homologue that β-cryptoxanthin or zeaxanthin increase.

[0109.0.10.10] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make each fine chemicals, particularly β-cryptoxanthin or zeaxanthin that the inventive method produced be optimized synthetic.

[0110.0.10.10] helps the inventive method and coding has the polypeptide of polypeptide of the present invention or the used polypeptide active of the used or of the present invention process of the inventive method (for example as Table II the 5th row, protein shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 or by as Table I the 5th be listed as, the protein of nucleic acid molecule encoding or its homologue are (as Table I or II the 7th row shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471)) nucleic acid molecule can from generally open database, determine.

[0111.0.0.10] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.10.10] the inventive method is the isolated nucleic acid sequences form, its coding have as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide active polypeptide or have as Table II the 5th and 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the polypeptide of peptide sequence, and give cryptoxanthin or the zeaxanthin level increases.

[0113.0.0.10] to [01200.0.10]: see that [0113.0.0.0] is to [0120.0.0.0]

[0120.1.10.10]: advantageously, the production bacterial strain of selecting in the inventive method is to be selected from following microorganism: green alga such as Spongioccoccum exentricum, Chlorella sorokiniana (Chlorella pyrenoidesa (pyrenoidosa) 7-11-05), perhaps fungi is as belonging to the fungi of colored otology (Daccrymycetaceae), perhaps non-photosynthetic bacterium such as methylotrophy bacterium, Flavobacterium, actinomycetes such as Streptomyces chrestomyceticus, mycobacterium such as Mycobacterium phlei (Mycobacterimphlei) or the red bacterium of pod membrane (Rhodobacter capsulatus).

[0121.0.10.10] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, peptide sequence or its functional homologue as herein described have the difference of one or more amino acid moleculars shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, giving the cryptoxanthin level shown in 103-106 and/or 468-471 are capable after the activity of peptide sequence increases, and is perhaps improving Table II the 5th or 7 row, giving the zeaxanthin level after the activity of peptide sequence shown in the 107th and/or 108 row increases.

[0122.0.0.10] to [0127.0.0.10]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.10.10] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in primer to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence or from as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence deutero-sequence produce.

[0129.0.10.10] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (sequence of polypeptide particularly of the present invention or the used polypeptide of the inventive method).The conservative region of polypeptide of the present invention or the used polypeptide of the inventive method is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in consensus sequence from described comparison.

[0130.0.10.10] can use degenerated primer to have the new protein fragment of aforementioned activity (giving zeaxanthin or cryptoxanthin increase as comprise this segmental protein expression or activity in raising after) by pcr amplification then.

[0131.0.0.10] to [0138.0.0.10]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.10.10] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with for the Table I the 5th of cryptoxanthin or 7 row, 103-106 and/or 468-471 are capable, preferred Table I B the 5th or 7 row, sequence shown in 103-106 and/or 468-471 are capable or for the Table I the 5th of zeaxanthin or 7 row, the 107th and/or 108 row, preferred Table I B the 5th or 7 row, sequence hybridization shown in the 107th and/or 108 row, and the coding expression has each fine chemicals, the i.e. active peptide of cryptoxanthin or zeaxanthin of increasing.

[0140.0.0.10] to [0146.0.0.10]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.10.10] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise one of the nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I B the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.10.10] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor contain following nucleotide sequence: with Table I the 5th or 7 row, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table I B the 5th or 7 row, nucleotide sequence or its portion homologous are at least about 30% shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471,35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases cryptoxanthin or zeaxanthin.

[0149.0.10.10] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table I B the 5th or 7 row, one of nucleotide sequence or the hybridization of its part shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferably in as literary composition, hybridize under the defined stringent condition, and coding have above-mentioned activity (as give cryptoxanthin or zeaxanthin increase) and randomly as Table II the 5th be listed as, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 5th or 7 row, the protein of protein active shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471.

[00149.1.10.10] randomly, in one embodiment, with Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I B the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein be known activity or be used for these protein of note.

[0150.0.10.10] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table I B the 5th or 7 row, the part of the coding region of one of nucleotide sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give cryptoxanthin or zeaxanthin increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in one of sequence sense strand, Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the right PCR of primer will produce as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 capable shown in fragment or its gene product of polynucleotide sequence.

[0151.0.0.10]: see [0151.0.0.0]

[0152.0.10.10] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coded polypeptide or its part, described polypeptide or its part comprise with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising cryptoxanthin (103-106 and/or 468-471 are capable) as be shown in the examples or zeaxanthin (the 107th and/or 108 row).

[0153.0.10.10] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein or its part have Table II the 3rd row for example as herein described, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the activity of polypeptide.

[0154.0.10.10] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.10] to [0156.0.0.10]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.10.10] the present invention relates in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, one of nucleotide sequence (with its part) shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 7th row, the polypeptide of consensus sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 or as Table II the 5th or 7 be listed as, the nucleic acid molecule of polypeptide or its function homologue shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment as Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in consensus sequence or as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in consensus sequence or as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet, in a preferred embodiment, nucleic acid molecule of the present invention do not comprise as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I A the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in nucleic acid molecule identical.

[0158.0.0.10] to [0160.0.0.10]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.10.10] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.10] sees [0162.0.0.0]

[0163.0.10.10] preferably, under stringent condition with Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given in biology or its part for example each fine chemicals quantity increase in the compartment of tissue, cell or cell after using activity of proteins).

[0164.0.0.10] sees [0164.0.0.0]

[0165.0.10.10] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.10] to [0167.0.0.10]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.10.10] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in aminoacid sequence at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in sequence identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, one of sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in sequence at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in sequence at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.10] to [0172.0.0.10]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.10.10] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:10237 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:10237 sequence before use.

[0174.0.0.10]: see [0174.0.0.0]

[0175.0.10.10] for example, the sequence that has 80% homology at protein level and SEQ ID NO:10238 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:10238 sequence.

[0176.0.10.10] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtains shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471,35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide has essentially identical character and discerns shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471.

[0177.0.10.10] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the functional equivalent that obtains of nucleotide sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471,35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471.

[0178.0.0.10] sees [0178.0.0.0]

[0179.0.10.10] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471) in introduce the replacement of one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, the nucleic acid molecule of homologue of protein sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in introduce sudden change in the encoding sequence of sequence.

[0180.0.0.10] to [0183.0.0.10]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.10.10] is employed to have as Table I the 5th or 7 row, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, perhaps come Table II the 5th or 7 row freely, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, the homologous compound of nucleotide sequence of sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.10.10] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in arbitrary sequence in other Nucleotide or the nucleotide sequence that do not show.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence identical.

The employed one or more nucleic acid molecule encodings of [0186.0.10.10] also preferred the inventive method comprise as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence identical.

[0187.0.10.10] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprise as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.10.10] still has the basic the enzyme activity of polypeptide of the present invention (being its active basic reduction that do not have) of giving each fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II the 3rd and 5 row, shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 and under the same conditions the polypeptide expressed activity is compared and basic is not reduced.In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence or by its homologue of forming.

[0189.0.10.10] as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the homologous compound of sequence, or deutero-as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the homologue of sequence also refer to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.10]: see [0190.0.0.0]

[0191.0.0.10] sees [0191.1.0.0]

[0192.0.0.10] to [0203.0.0.10]: see that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.10.10] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide or its segmental nucleic acid molecule, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly β-cryptoxanthin (103-106 and/or 468-471 are capable) or zeaxanthin (the 107th and/or 108 row) quantity increases;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in nucleic acid molecule or its segmental nucleic acid molecule, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprise by use as Table III the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in primer or primer nucleic acid molecule that amplification from cDNA library or genomic library is obtained, and in biological or its part, give each fine chemicals, i.e. the increase of β-cryptoxanthin (103-106 and/or 468-471 are capable) or zeaxanthin (the 107th and/or 108 row) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in consensus sequence and in biological or its part, give each fine chemicals, the i.e. nucleic acid molecule that increases of β-cryptoxanthin (103-106 and/or 468-471 are capable) or zeaxanthin (the 107th and/or 108 row) quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be the nucleic acid molecule that β-cryptoxanthin (103-106 and/or 468-471 are capable) or zeaxanthin (the 107th and/or 108 row) quantity increases, described peptide coding as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table II B the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the structural domain of polypeptide; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in nucleic acid molecule coding (optimized encoding is mature form at least) as Table II the 5th or 7 row, capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred Table II B the 7th row, at least the 15nt of nucleic acid molecule of polypeptide shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby, the nucleic acid molecule of preferred (a) to (I) by one or more Nucleotide be different from as Table I A the 5th or 7 row, 1-5 is capable and/or the sequence of 334-338 shown in capable.In one embodiment, nucleic acid molecule not by Table I A or IB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence form.In one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in peptide sequence.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence at least 30%, 40%, 50% or 60% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In other embodiments, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in peptide sequence.Therefore, in one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in nucleic acid molecule have at least one or more residues different.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide have the different polypeptide of one or more amino acid at least.In another embodiment, Table I A or IB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in nucleic acid molecule do not encode as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the protein of sequence.Therefore, in one embodiment, by nucleotide sequence (a)-(I) encoded protein matter not by Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence form.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in protein sequence at least 30%, 40%, 50% or 60% identical and with Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% identical.

[0205.0.0.10] to [0206.0.0.10]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.10.10] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of carotenoid metabolism, xenthophylls metabolism, astaxanthin metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.10] to [0226.0.0.10]: see that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.10.10] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 sequence or derivatives thereofs mentioned of row, can advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one xenthophylls biosynthetic pathway in biological (as plant or microorganism), for example cryptoxanthin or zeaxanthin gene, one of above-mentioned this pathway gene for example, perhaps for example astaxanthin synthetic gene or another provitamin A gene or another carotenoid gene.Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.10.10] therefore cultivated to cross simultaneously in another embodiment of the present invention and expressed coding and xenthophylls metabolism (particularly β-cryptoxanthin, zeaxanthin, astaxanthin or xenthophylls are synthetic) at least one nucleic acid of related protein or the biology of a gene.

[0229.0.10.10] can make up the sequence of other favourable nucleotide sequences of expression for other gene of coding carotenoid biosynthetic pathway with sequence that present method is used and/or aforementioned biosynthesis gene, for example (it is the important control point of flux adjusting to phytoene synthase (Psy), see Fraser etc., 2002), phytoene desaturase (Pds), z-carotene desaturase, above-mentioned enzyme (seeing the preface part of application) for example hydroxylase such as β-Hu Luobusu hydroxylase (US 6,214,575), (US 6 for ketolase or cyclase such as beta cyclase, 232,530) or oxygenase such as US 6,218, β-C4-oxygenase or its homologue described in 599, (US 6 for the astaxanthin synthase, 365,386), perhaps US 6,150, other gene of describing in 130.These genes cause the synthetic increase of essential carotenoid, particularly xenthophylls.

[0230.0.0.10] sees [230.0.0.0]

[0231.0.10.10] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degrading maize xanthin or cryptoxanthin simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.The known synthetic quantity that can increase cryptoxanthin and zeaxanthin that in biology, particularly plant, suppresses xenthophylls from carotene of those skilled in the art.In one embodiment, the astaxanthin level in the biology will be enhanced.Therefore, the enzyme of degraded astaxanthin, rather than the catalysis astaxanthin is weakened from zeaxanthin or cryptoxanthin synthetic enzyme.

[0232.0.0.10] to [0276.0.0.10]: see that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.10.10] can separate each fine chemicals that produces by the method that the technician is familiar with from biology, for example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.Fine chemicals and other xenthophylls of producing by this process can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing plant part or extract realization.

[0278.0.0.10] to [0282.0.0.10]: see that [0278.0.0.0] is to [0282.0.0.0]

[0283.0.10.10] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II the 3rd row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in protein, perhaps as Table II the 5th or 7 row, the antibody of polypeptide or its antigen part shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, it can comprise above-mentioned sequence or by its polypeptide of forming by the standard technique utilization, and polypeptide for example of the present invention or its fragment (being polypeptide of the present invention) produce.Preferably specificity in conjunction with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the monoclonal antibody of polypeptide.

[0284.0.0.10] sees [0284.0.0.0]

[0285.0.10.10] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in sequence by as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.10.10] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence or by its polypeptide of forming shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the polypeptide of consensus sequence (each row).

[0287.0.0.10] to [0289.0.0.10]: see that [0287.0.0.0] is to [0289.0.0.0]

[00290.0.10.10] uses Vector NTI Suite 8.0, (InforMax ^TM, Invitrogen ^TMLifescience software, U.S.Main Office, 7305Executive Way, Frederick, MD21704, U.S.) assembly AlignX software (on September 25th, 2002) and use following setting to compare: for the pairing comparison: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Right for multiple ratio: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Breach separates the point penalty scope: 8; Residue is replaced matrix: blosum62; Hydrophilic residue: G P S N D Q E K R; Conversion weight: 0.5; Total calculation options: total residue mark: 0.9.Also selected to set in advance in order to compare conserved amino acid.

[0291.0.10.10] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in sequence, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in sequence.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence form.

[0292.0.0.10] sees [0292.0.0.0]

[0293.0.10.10] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in other sequence of sequence phase region.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence form.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help Table I A or IB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the coded sequence of nucleic acid molecule form.

[0294.0.10.10] in one embodiment, the present invention relates to have as Table II the 3rd row, the polypeptide of activity of proteins shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in sequence.

[0295.0.0.10] to [0297.0.0.10]: see that [0295.0.0.0] is to [0297.0.0.0]

The chemical of [00297.1.0.10] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, 103-108 or 468-471 capable shown in the activity of polypeptide and/or the polypeptide of aminoacid sequence.[0298.0.10.10] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in the identical aminoacid sequence of sequence.

[0299.0.10.10] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in one of nucleotide sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.10.10] is therefore, and be described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 capable shown in sequence different.Therefore, this polypeptide contain with as Table II A or IIB the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the complete amino acid sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.10] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.10.10] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in aminoacid sequence or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.10] sees [0303.0.0.0]

[0304.0.10.10] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.10] to [0306.0.0.10]: see that [0305.0.0.0] is to [0306.0.0.0]

[0306.1.0.10] preferably, compound is the composition that comprises each fine chemicals of pure basically cryptoxanthin or zeaxanthin or recovery or isolating cryptoxanthin or zeaxanthin, particularly free form or protein bound form.

[0307.0.0.10] to [0308.0.0.10]: see that [0307.0.0.0] is to [0308.0.0.0]

[0309.0.10.10] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, 103-106 and/or 468-471 capable or the 107th and/or 108 the row shown in protein be meant polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " non-polypeptide of the present invention " or " other polypeptide " polypeptide of being meant aminoacid sequence in capable or the 107th and/or 108 row of 103-106 and/or 468-471 with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, polypeptide shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, proteinic and shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471 from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or " the non-polypeptide of the present invention " or " other polypeptide " of the 107th and/or 108 row are not given in biology or its part each fine chemicals and increased.

[0310.0.0.10] to [0334.0.0.10]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.10.10] confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in nucleotide sequence and/or the expression of its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.10] to [0342.0.0.10]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.10.10] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.10] to [0361.0.0.10]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.10.10] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, polypeptide shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471, for example coding has protein such as Table II the 3rd row, the polypeptide of polypeptide active shown in capable or the 107th and/or 108 row of 103-106 and/or 468-471) nucleic acid molecule.

Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.In one embodiment, have as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide (for example have as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

[0363.0.0.10] sees [0363.0.0.0]

[0364.0.10.10] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---for example naturally occurring coding as Table II the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide of the present invention gene promotor with accordingly as Table I the 3rd row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the combination of protein coding sequence---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.10] to [0373.0.0.10]: see that [0365.0.0.0] is to [0373.0.0.0]

[0374.0.10.10] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the xenthophylls that produces in the inventive method, particularly each fine chemicals also can be from plant with free lutein, particularly each fine chemicals or be bonded to compound or the isolated in form of part (moiety) (for example glucosides such as bioside).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.10] to [0376.0.0.10]: see that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.10.10] therefore the invention still further relates to the method according to this invention, separated the composition that contains carotenoid, particularly xenthophylls that is produced thus.In one embodiment, separated each fine chemicals that is produced.

[0378.0.10.10] can separate by this way and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than xenthophylls, particularly each fine chemicals of producing in the method for 90%.As required, the xenthophylls (composition that for example comprises xenthophylls) of resulting recovery, isolated or purified can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.10.10] in one embodiment, xenthophylls is the mixture of each fine chemicals.In one embodiment, xenthophylls is each fine chemicals.In one embodiment, xenthophylls is the mixture of each fine chemicals and astaxanthin.

The suitable synthetic parent material of the xenthophylls that [0380.0.10.10] obtains in the method for the invention, particularly each fine chemicals as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises the composition that comprises carotenoid, particularly xenthophylls that separation produces or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention be the carotenoid that produces of the inventive method or xenthophylls or genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine purposes or be used to produce the purposes of astaxanthin, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

[0381.0.0.10] to [0382.0.0.10]: see that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.10.10]./.

[0384.0.0.10] sees [0384.0.0.0]

The fermented liquid that [0385.0.10.10] obtains with this kind approach, particularly contain fermented liquid with other carotenoid, particularly other xenthophylls such as astaxanthin blended zeaxanthin or β-cryptoxanthin, perhaps contain and comprise and the microorganism of other carotenoid, particularly other xenthophylls such as astaxanthin blended zeaxanthin or β-cryptoxanthin or the fermented liquid of microorganism part (as plastid), usually dry matter content accounts for 1 to 70% of weight, preferred 7.5 to 25%.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means that the concentration that can utilize sugar in the fermention medium during this period of time remains on or be reduced to 0 to 10g/l, preferred 0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

Because carotenoid is usually located at film or plastid, therefore in one embodiment, when by separation method such as centrifugal, filtration, decant, condense/flocculate or the combination of these methods from the fermented liquid kind completely or partially during the separating bio amount, avoiding cell is favourable by filtration.The biomass of doing directly can be added in the animal-feed, as long as the enough height and do not have toxic chemical of the concentration of carotenoid.Consider the unstable of carotenoid, drying conditions such as spraying drying or expansion drying are wanted gentle and will be avoided oxidation and suitable/anti-isomerization.Can add antioxidant, for example BHT, ethoxyquinoline or other antioxidant.Under the concentration of carotenoid situation to be diluted, can use solvent extraction to separate, for example use alcohol, ether or other organic solvent (as methyl alcohol, ethanol, acetone, potassium hydroxide alcohol, phenol glycerine, liquefied phenol) or with acid or alkali such as trichoroacetic acid(TCA) or potassium hydroxide.Can find the multiple favorable method and the technology of separation carotenoid, particularly xenthophylls, particularly zeaxanthin or cryptoxanthin in current state of the art.Under the situation of using phenol, phenol can remove with ether and water extraction, and the exsiccant elutriant comprises biomass carotenoid mixture.

[0386.0.10.10] therefore can also be further purified the carotenoid, the particularly xenthophylls that produce according to the present invention.For this reason, the composition that will contain product, the lipid extracts fraction total or part that for example with an organic solvent obtains is as mentioned above carried out saponification (to remove triacylglycerol), distributes (distributing) (from more separating nonpolar epiphase the low phasic property derivative of polar) and separate (by for example open column chromatography or HPLC) between hexane/methanol, and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.10] to [0392.0.0.10]: see that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.10.10] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row, preferred Table I B the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the nucleic acid molecule nucleic acid molecule of under lax stringent condition, hybridizing, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.10] to [0398.0.0.10]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.10.10] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving each fine chemicals generation raising under these conditions.

[00399.1.0.10] it is contemplated that by for example seek to the resistance of blocking each fine chemicals synthetic medicine and observe this effect whether depend on as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.10] to [0416.0.0.10]: see that [0400.0.0.0] is to [0416.0.0.0]

[0417.0.10.10] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of carotenoid biosynthetic pathway is had resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block carotenoid in the described biology (particularly each fine chemicals) synthetic inhibitor.

The example of carotenoid synthetic inhibitor or weedicide can be divided into two groups in biological for example microorganism of blocking-up or the plant.First group comprises the early stage intermediate product that causes in this approach, the inhibitor of particularly colourless plant alkene accumulation, for example pentanoic.Other inhibitor is preferably blocked the late phase reaction in this approach, particularly cyclization of lycopene.Inhibitor is nicotine, 2-(4-chlorobenzene sulfo-)-triethylamine and other replacement amine and nitrogen heterocyclic ring base, for example imidazoles.

Biological antioxidant is coerced, the particularly damage of singlet oxygen because xenthophylls can be protected, and the level that therefore increases each fine chemicals can the anti-weedicide that causes producing oxygenated compound (for example singlet oxygen) of protective plant.For example, the inhibition to a kind of important enzyme proporphyrinogen oxidase (Protox) in chlorophyll and the protoheme biosynthesizing causes the loss of Chlorophylls and Carotenoids and causes producing the seepage film; The destruction of film is owing to the generation (weedicide for other typical photosynthetic inhibitor that becomes second nature also has report) of free oxygen free radical.

Therefore, in one embodiment, the increase of each fine chemicals level is used for the anti-weedicide that destroys film (because generation of free oxygen free radical) of protective plant.

Producing the inhibitor of oxidative stress or the example of weedicide is aryl triazoles (aryl triazion), for example sulfentrazone, fluorine ketazolam grass; Perhaps diphenyl ether, for example acifluorfen, lactofen or Oxyfluorfen; Perhaps N-phenylphthalimide, for example imide phenoxy acetic acid Huo Fu oxazinone; Substituted urea class, for example fluometuron, Metribuzin, Diuron Tech or methoxydiuron; Perhaps triazines, for example G-30027, prometryn, ametryn, sencorex, prometon, simazine or six piperazines with; Perhaps uracil, for example bromacil or terbacil.

The carotenoid inhibitor is for example pyridine and pyridazinone, for example monometflurazone, fluorine humulone or dithiopyr.Therefore, in one embodiment, the present invention relates to improve the purposes according to each fine chemicals of the present invention, it is used for the injury that protective plant is avoided carotenoid inhibitor such as pyridine and pyridazinone.

[0418.0.0.10] to [0423.0.0.10]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.10.10] therefore, nucleic acid of the present invention or the inventive method nucleic acid molecule used therefor, polypeptide of the present invention or the used polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other carotenoid, particularly other xenthophylls such as astaxanthin.

Therefore, nucleic acid of the present invention or the inventive method nucleic acid molecule used therefor or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention or the used polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0424.1.0.10] the present invention relates to the purposes that antagonist of the present invention, plant of the present invention or its part, microorganism of the present invention or host cell or its part are used for production cosmetic compositions or pharmaceutical composition in other embodiments.This composition has anti-oxidant activity, the light protection is active, tanning is active, but for example it is used for the treatment of high-level cholesterol and/or fat with pharmaceutically acceptable carrier or cosmetic carrier combinations; Be used for protection, treat or cure above-mentioned disease (for example retinal diseases, height

Hypercholesterolemia, hyperlipidemia and arteriosclerosis); Perhaps be used for cleaning, conditioning and/or handle skin.

Xenthophylls can be used as the stablizer of other color sensitivity or oxygen sensitive compound.

[0425.0.0.10] to [0434.0.0.10]: see that [0425.0.0.0] is to [0434.0.0.0]

[0435.0.10.10] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.10.10] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement green alga (Spongiococcum sp (for example Spongiococcumexentricum) for example, Chlorella certain (Chlorella sp.)), yeast belong, genus mortierella, the mutagenesis in vivo of Escherichia and above-mentioned other genus is beneficial to produce xenthophylls.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli andSalmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0436.1.0.10] sees [0436.1.0.0]

[0437.0.10.10] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.10.10] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.10.10] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.10.10] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad 50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.10] to [0443.0.0.10] sees that [0441.0.0.0] is to [0443.0.0.0]

[0444.0.10.10] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.10.10] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.10] to [0454.0.0.10]: see that [0446.0.0.0] is to [0454.0.0.0]

[0455.0.10.10] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example lipid acid) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being lipid or lipid acid) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

Except said process, as (1999) Proc.Natl.Acad.Sci.USA96 (22) such as Cahoon: from vegetable material, extract plant lipid as described in (1986) Analytic Biochemistry152:141-145 such as 12935-12940 and Browse.The qualitative and quantitative analysis of lipid is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:Oily Press (Oily Press Lipid Library; 2); Christie, William W., GasChromatography and Lipids.A Practical Guide-Ayr, Scotland:Oily Press, 1989, Repr.1992, IX, 307 pp. (Oily Press Lipid Library; 1); " Progress inLipid Research, Oxford:Pergamon Press, 1 (1952)-16 (1977), exercise question is Progress in the Chemistry of Fats and Other Lipids CODEN.

[0456.0.0.10]: see [0456.0.0.0]

[0457.0.10.10] embodiment 9: the purifying of xenthophylls and carotenoid content are measured

[0458.0.10.10] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS or TLC can clearly detect the existence of xenthophylls to the biology analysis of recombinating.

It is biological to use the following step to analyze, as the total xenthophylls that produces in the inventive method used yeast: by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000 W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 4022S1V diluter with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.10ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, Hewlett Packard 1100 HPLC have been used, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Technologies, can be from Ultra Scientific Inc., 250 Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6x250mm[Waters, 34 Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

For selected wild-type and the mutant sample of from the screening step, identifying, measure carotenoid compositions.The petal sample can be stored in-80 ℃ of refrigerators until identifying mutant.Can sample is moving dried, and the exsiccant tissue can be stored in-80 ℃ of guidance preparation analyses in the presence of argon.

Extraction step can carry out under ruddiness.Dry petal is ground and sieve with the No.40 mesh sieve.Accurately weighing grinds the weight of sample and is transferred in the red volumetric flask of 100ml.In sample, add 500 μ l H ₂O was with mixture vortex vibration 1 minute.Adding 30ml extracts solvent (10ml hexane+7ml acetone+6ml raw spirit+7ml toluene) and bottle was vibrated 10 minutes down at 160 rev/mins.

For saponification reaction, in bottle, add 2ml 40%KOH methanol solution, the vortex vibration is 1 minute then.Bottle is placed 56 ℃ of water-baths 20 minutes.Connect condenser to prevent solvent loss.Use the condenser that is connected in the dark with sample cooling 1 hour.After the cooling, add the 30ml hexane, and bottle was vibrated 10 minutes with 160 rev/mins.

Diluted sample after will vibrating with 10% metabisulfite solution is to 100ml, and thermal agitation 1 minute.Sample was placed the dark place at least 30 minutes.Take out the aliquots containig of 35ml mutually from the upper strata of about 50ml and be transferred to the sample cup.In bottle, add the 30ml hexane again, then with 160 rev/mins of vibrations 10 minutes.After about 1 hour, the upper strata is mixed mutually.Analyze for HPLC, the 10ml aliquots containig is dry and be stored in-80 ℃ in the presence of argon under nitrogen.

HPLC equipment is Alliance 2690, it is equipped with the cooling self-actuated sampler, twists well heater Waters Photodiode Array 996 detectors (Waters Corp., 34 Maple StreetMilford, MA 01757). separation can (3m 2.0x150mm) realizes with having YMC30 post with the Material card sleeve column.Standard can be from ICC Indorespective fine chemicalsSomerville, New Jersey 088876 and DHI-Water﹠amp; Environment, DK-2970Horsholm, Denmark obtains.

Exsiccant mutant sample can be resuspended in tetrahydrofuran (THF) and the methyl alcohol to cumulative volume 200l and filter, and contrast can additionally not concentrate.Carotenoid can separate with gradient method.Initial gradient condition can be 90% methyl alcohol: 5% water: 5% methyl tertiary butyl ether, flow velocity 0.4ml/min.From 0 to 15 minute, moving phase can become 80 methyl alcohol from initial condition: 5 water: 15 methyl tertiary butyl ethers from 15 to 60 minutes, are changed to 20 methyl alcohol: 5 water: 75 methyl tertiary butyl ethers.For later 10 minutes, the moving phase condition can be back to initial condition, and with column equilibration 10 minutes.Column temperature can be maintained at 27 ℃ and flow velocity and maintain 0.4ml/min.Injection 10l.Most peaks can 450nm measure and can from 286,348,400 and 472nm extract passage and add extra area.

[0459.0.10.10] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified xenthophylls.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.10.10] sees [0460.0.0.0]

[0461.0.10.10] embodiment 10: clone SEQ ID NO:10215 is used for expressing plant

[0462.0.0.10] sees [0462.0.0.0]

[0463.0.10.10] passes through pcr amplification SEQ ID NO:10215 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.10] to [0466.0.0.10]: see that [0464.0.0.0] is to [0466.0.0.0]

[0466.1.0.10] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.10.10] selects following primer sequence for genes of SEQ ID NO:10215:

I) forward primer (SEQ ID NO:10235)

atgcgccctc?ttattttatc?gatttt

Ii) reverse primer (SEQ ID NO:10236)

ttatggtgcg?ggtttaagaa?acgtc

[0468.0.0.10] to [0479.0.0.10]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.10.10] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:10215

[0481.0.0.10] to [0513.0.0.10]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.10.10] selects as another kind of, as described (Deli, J.﹠amp; Molnar, P., Paprikacarotenoids:Analysis, isolation, structure elucidation.Curr.Org.Chem.6,1197-1219 (2004) or Fraser, P.D., Pinto, M.E., Holloway, D.E.﹠amp; Bramley, P.M.Technical advance:application of high-performance liquidchromatography with photodiode array detection to the metabolicprofiling of plant isoprenoids.Plant J.24,551-558 (2000)) detect xenthophylls.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??MAX
					??YCL040W	Cryptoxanthin	??LC	??1.36	??1.51
??b0986	Cryptoxanthin	??LC	??1.33	??1.40
					??b3684	Cryptoxanthin	??LC	??1.35	??1.67
??b4401	Cryptoxanthin	??LC	??1.48	??1.60
					??b3926	Cryptoxanthin	??LC	??1.33	??1.33
??b0851	Cryptoxanthin	??LC	??1.57	??1.76

??b2211	Cryptoxanthin	??LC	??1.05	??1.26
					??b0050	Cryptoxanthin	??LC	??1.42	??1.84
??YHR055C	Zeaxanthin	??LC	??1.05	??1.38
					??b2699	Zeaxanthin	??LC	??1.25	??1.48

[0515.0.10.10] to [0552.0.0.10]: see that [0515.0.0.0] to [0552.0.0.0] comprises

[0530.1.0.0] to [0530.6.0.0] and [0552.1.0.0] and [0552.2.0.0]

[0553.0.10.10]

1. produce the method for xenthophylls, it comprises:

(a) improve in non-human being or its one or more parts or produce one or more as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation xenthophylls.

2. produce the method for xenthophylls, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part xenthophylls quantity to be increased;

B) contain just like Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the nucleic acid molecule of nucleic acid molecule;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of xenthophylls quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of xenthophylls quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that xenthophylls quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that each fine chemicals quantity of xenthophylls increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that xenthophylls quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in consensus sequence polypeptide and give biology or its part in each fine chemicals quantity of xenthophylls increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give each fine chemicals quantity increase of xenthophylls in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded xenthophylls.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded xenthophylls of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part xenthophylls quantity to be increased;

B) contain just like Table I the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in the nucleic acid molecule of nucleic acid molecule;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of xenthophylls quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of xenthophylls quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that xenthophylls quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that each fine chemicals quantity of xenthophylls increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that xenthophylls quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in consensus sequence polypeptide and give biology or its part in each fine chemicals quantity of xenthophylls increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give each fine chemicals quantity increase of xenthophylls in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 the row shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding as claim 6 described in, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 103-106 and/or 468-471 is capable or the 107th and/or 108 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in xenthophylls quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part xenthophylls quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps lutein levels or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the lutein levels that will measure or expression of polypeptides level and standard xenthophylls or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the xenthophylls generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of xenthophylls quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of xenthophylls quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the xenthophylls generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that xenthophylls increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce xenthophylls;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) lutein levels in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give lutein levels raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the xenthophylls generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that xenthophylls quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce xenthophylls;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) lutein levels in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give lutein levels raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that xenthophylls increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control lutein levels at biology.

25. makeup, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the polypeptide of the nucleic acid molecule of any one described method, claim 6, claim 14, claim 7 among the claim 1-5, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein xenthophylls is respectively β-cryptoxanthin or zeaxanthin.

27. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make, and plant is anti-oxidant coerces.

28. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make, and plant is anti-causes oxidative stress or suppress carotenoid synthetic weedicide.

29. the purposes of the results material of the agonist of identifying according to claim 17, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12, it is used to produce makeup or pharmaceutical composition.

[0554.0.0.10] summary: see [0554.0.0.0]

Produce the method for fine chemicals

Above [0001.0.0.12] sees for disclosing of this paragraph [0001.0.0.0].

[0002.0.12.12] sterol is the essential natural compounds of dependence that all eukaryotic cells all need in order to finish its life cycle.In animal, cholesterol generally is important sterol, and ergosterol is important sterol in fungi.Plant produces the sterol that a class is called plant sterol.Plant sterol is the natural component of many vegetables and cereal.Plant sterol and plant dihydrotestosterone contained in term " plant sterol ".Plant sterol is that the submember as plant oil is present in the natural occuring article matter in the diet.The structure of these plant sterols and the structural similitude of cholesterol have extra methyl or ethyl and two key in side chain.Saturated plant sterol is called stanols, does not have two keys in ring structure.Therefore plant sterol (comprising plant sterol and stanols) is vegetation foodstuff particularly plant oil, seed and nut, cereal and a beans, the particularly natural component of edible plants oils such as wunflower seed oil, the natural component in the human diet just.

High density cholesterol in [0003.0.12.12] serum, promptly hypercholesterolemia is well-known coronary heart disease (CHD) Hazard Factor.Can reduce blood cholesterol levels by following diet, these diet are low saturated fatty, high poly unsaturated fatty acids and low-cholesterol.Although obtained suitable achievement from the viewpoint of knowledge and education, the human consumer finds still to be difficult to follow healthy dietary recommendation.Plant sterol and phytostanols can both reduce blood plasma summation low-density lipoprotein (LDL) cholesterol effectively, and this realizes by suppressing to absorb cholesterol from small intestine.Plant sterol reduces the characteristic of plasma cholesterol and just knows (Pollak, Circulation, 7,702-706,1953) since nineteen fifties.Plant sterol is as the reducing cholesterol agent, at first with free form (Pollak and Kritchevsky, Sitosterol, In:Monographs onAherosclerosis.Clarkson TB, Kritchevsky D, Pollak OJ, eds.New York, Basel, Karger 1981; 1-219), and at present mainly be used as esterification plant sterol (Katan etc., Mayo Clin Proc 2003; 78:965-978).The consumption of plant sterol and phytostanols reduces blood cholesterol levels by inhibition from the cholesterol that small intestine absorbs diet and endogenous generation, and plant sterol/stanols self is difficult to absorb very much.This restraining effect is relevant with the similarity of the physicochemical property of plant sterol and stanols and cholesterol.Shown that in a large amount of human research plant sterol and phytostanols can health risks, do not had deleterious effect.Plant sterol and phytostanols show or even also do not have toxicity in the high dosage level, and its gastrointestinal absorption effect is lower.

The abundantest sterol of [0004.0.12.12] vascular plant is campesterol, Sitosterol and Stigmasterol, and they all contain two keys between the carbon atom at position 5 and 6 places, and classify as Δ-5 sterol.As illustration naturally occurring Δ-5 plant sterol is isofucosterol, Sitosterol, Stigmasterol, campesterol, cholesterol and Neospongosterol,dihydro-.As the naturally occurring non-Δ-5 of illustration plant sterol is cycloartenol, 24-methylene radical cycloartenol, cycloeucalenol and obtusifoliol.The ratio of Δ in the plant-5/ non-Δ-5 sterol is the important factor relevant with the insect pest resistance.Insect can not the de novo synthesis steroid nucleus and depends on the external source sterol in its food source so that produce essential sterid.Particularly, insect needs external source Δ-5 sterol.By embodiment, the Δ that external source provides-5 sterol is essential for the ecdysteroid hormone-ecdysteroid that produces control reproduction and growth.See Costet etc. for example, Proc.Natl.Acad.Sci.USA, 84:643 (1987) and Corio-Costet etc., Archivesof Insect Biochem.Physiol., 11:47 (1989).

Described among [0005.0.12.12] US 20020148006 and the WO 98/45457 and regulated plant sterol by the dna sequence dna that uses first enzyme of coding and form, and/or improved the nutritive value of plant so that give resistance to insect, nematode, fungi and/or environmental stress; Wherein first enzyme is in conjunction with first sterol and be preferably selected from S-adenosine-L-methionine(Met)-24 (25)-sterol methyltransgerase, C-4 demethylase, cycloeucalenol-obtusifoliol-isomerase, 14--demethylase, 8-7-isomerase, 7-C-5-desaturase and 24, the 25-reductase enzyme, and produce second sterol and cause 3 ' non-translational region of polyadenylation at RNA 3 ' end.WO 93/16187 discloses and comprised the early stage gene of one or more involved in plant sterol biosynthesizing in its genome, be preferably the coding Mevalonic kinase gene, new plant.US 5,306, and 862, US 5,589,619, US 5,365, and 017, US5,349,126 and US 20030150008 method that increases sterol (and squalene) accumulation plant has been described, this method based on improving the HMG-CoA reductase activity so that improve the insect-resistance of transgenic plant.WO 97/48793 discloses the C-14 sterol reductase enzyme polypeptide that is used for the genetic manipulation of plant sterol biosynthetic pathway.US 20040172680 discloses the level that the gene that will express SMT1 (sterol methyltransgerase) is used for improving the plant seed sterol.The dna sequence dna that separates the coding sterol methyltransgerase that obtains from Zea mays is disclosed in WO 00/08190.Bouvier-Nav etc. have described two sterol methyltransgerases (SMT) family at Eur.J.Biochem.256 among the 88-96 (1988), first (SMT1) for being applied to cycloartenol, second (SMT2) is applied to 24-methylene radical lophenol.Schaller etc. (plant Physiology (1998) 118:461-169) describe the SMT2 that belongs to from mouse ear mustard and cross and be expressed in the change that tobacco causes 24-base cholesterol and Sitosterol ratio in the tobacco leaf.US 6,723,837 and US 20040199940 protein and the segmental nucleic acid molecule of coding and sterol and plant sterol metabolism related protein are disclosed, and operated and make it contain at least a sterol of improving the standard or cross expressing or the cell of plant sterol compound.Protein or fragment are selected from HES1, HMGCoA reductase enzyme, squalene synthase, cycloartenol synthase, SMTI, SMTII and UPC, be preferably selected from and comprise oxygen sterol conjugated protein consensus sequence-E (K, Q) xSH (H, R) PPx (S, T, A, C, F) the KES1/HES1/OSH1 family oxygen sterol of A is in conjunction with (OSBP) protein.Class protein, oxygen sterol conjugated protein has carried out reporting (it is all integrated as a reference for Jiang etc., yeast 10:341-353 (1994)) in people and yeast.These protein have been reported the ergosterol level (Jiang etc., yeast 10:341-353 (1994)) that can regulate in the yeast.Particularly, Jiang etc. have reported three gene KES1, HES1 and OSH1, and their codings contain the protein of aerobic sterol calmodulin binding domain CaM.

[0006.0.12.12] has the transgenic plant that change the sterol spectrum and can be used in the sitotherapy of setting up cholesterol processing and cardiovascular disorder prevention.The plant sterol spectrum that changes also can cause insect-resistance.

[0007.0.12.12] although people consume plant sterol every day in its normal diet, quantity not sufficient is to have the effect of significant reduction blood cholesterol levels.According to the food article that is consumed, the absorption of plant sterol is different between different groups, and still average daily westerner's diet is reported as and contains these sterols of 150-300mg (de Vries etc., J Food Comp Anal 1997; 19:115-141;

Deng, Inborn errors in bile acid biosynthesis and storage ofsterols other than cholesterol.In:The Metabolic and Molecular Bases ofInherited Disease.Scriver CS, Beaudet AL, Sly WS, Valle D, eds.NewYork, McGraw-Hill 2001; 2961-2988).In order to obtain the reducing cholesterol effect, need to consume the plant sterol (Hendriks etc., European Journal of ClinicalNutrition, 53,319-327,1999) in about 1 gram/sky.

[0008.0.12.12] plant sterol was found with low-level natural being present in the vegetable food product.The concentrated of food such as the oleomargarine that contains plant sterol and stanols is in one of recent development direction aspect the functional food, so that strengthen the reducing cholesterol ability of conventional food product.It may be the effective ways that reduce summation LDL cholesterol levels that extra plant sterol is mixed diet.Non-esterified plant sterol can be used as new food composition in the following:

(a) bakery product and cereal (for example breakfast cereal food, breakfast bar);

(b) milk-product such as low fat and fat reducing liquid milk, low fat and fat reducing yogourt and yogourt goods, and the dessert of every day;

(c) not carbonated soft drink such as low fat and fat reducing soybean drink and low fat and fat reducing soy acid cheese;

(d) meat product or edible-fat and oils (for example mayonnaise, seasonings, salad-dressing)

(e) oleomargarine; With

(f) meal liniment (table spreads) or dietary fat.

[0009.0.12.12] when using oils to carry out normal refining, plant sterol is by extracting section.Need 2500 tons of vegetables of refining to produce 1 ton of plant sterol according to estimates.Phytostanols obtains by the hydrogenization of plant sterol.

Another source of [0010.0.12.12] plant sterol is a Tall oil, derives from from timber to produce the course of processing of paper, and produces 1 ton of about 2500 tons of pine tree of plant sterol needs.Tall oil also contains a high proportion of phytostanols (being mainly the b-sitostanol) than vegetables oil.

[0011.0.12.12] as mentioned above, plant sterol is used for a large amount of different application, for example is used for makeup, medicine and feed and food.Therefore, the quality of raising food and animal-feed is the vital task of food and fodder industry.Particularly advantageous for food and animal-feed quality is the sterol spectrum in the balanced diet as far as possible.

[0012.0.12.12] therefore still exists great demand new and more suitable gene, the described biosynthetic enzyme of genes encoding involved in plant sterol and can produce them and not form unnecessary byproduct in the particular industry scale.In the gene of selecting the biosynthesizing instrumentality, above-mentioned two features are particularly importants.On the one hand, but still need to obtain the development of the highest intrinsic energy plant sterol, on the other hand, produce byproduct in process of production as few as possible.

Above [0013.0.0.12] sees for disclosing of this paragraph [0013.0.0.0]

[0014.0.12.12] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is a plant sterol.Term " plant sterol " comprises sterol of plant and the stanols of plant.Therefore, in the present invention, term " fine chemicals " is meant " " plant sterol " as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of plant sterol and phytostanols, preferred Sitosterolum, campesterol and/or Stigmasterol as used herein.

[0015.0.12.12] in one embodiment, term " fine chemicals " meaning is meant plant sterol, the sterol of plant and the stanols of plant.In whole specification sheets, term " fine chemicals " meaning is meant free form or is bonded to plant sterol and ester, thioesters or the sterol of other compound form.For purpose of description, the term sterol refers to free sterol and the sterol of puting together, for example the 3-hydroxyl by fatty acid chain or phenylic acid esterification to produce sterol ester/stanol ester.

As used herein, term " plant sterol " without stint comprises all plant sterols, for example Sitosterol, campesterol, Stigmasterol, brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol, corresponding stanols and all natural or synthesized form and derivative thereof comprise isomer.Required understanding to plant sterol, comprises that promptly side chain modifies also to fall within the scope of the invention.All that derivative summary is become " conjugate ".In preferred embodiments, term " fine chemicals " or term " plant sterol " or term " each fine chemicals " meaning is meant at least a being selected from " Sitosterolum, sitostanol, Stigmasterol, brassicasterol, campestanol, isofucosterol and campesterol ", preferred " Sitosterolum, campesterol and/or Stigmasterol ", most preferably the chemical compound plant sterol and the phytostanols of " Sitosterolum and/or campesterol ".The ester that equally preferably has the sterol of C10-24 lipid acid.

The plant sterol content that increases is often referred to and improves total plant sterol content.Yet, the plant sterol content that increases also refers in particular to have the active above-claimed cpd of the plant sterol improvement content of (" Sitosterolum, sitostanol, Stigmasterol, brassicasterol, campestanol, isofucosterol and campesterol "), and does not need to increase total plant sterol content.

[0016.0.12.12] therefore the present invention relates to produce the method for plant sterol, and it comprises:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YER156C, YKR057W, YOR084W, b0019, b0421, b2699, b0050, b0161 and/or b4129 protein and/or YER156C, YER173W, YKR057W, YOR044W, YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or b2822 protein and its one or more parts of in a non-human being in and

(b) in allowing described biology, produce fine chemicals, for example plant sterol, plant sterol and stanols, preferred Sitosterolum, sitostanol, Stigmasterol, brassicasterol, campestanol, isofucosterol and campesterol, more preferably Sitosterolum, campesterol and/or Stigmasterol, most preferably Sitosterolum and/or campesterol or comprise under the condition of fine chemicals of plant sterol and make biological growth.

Therefore, the present invention relates to produce the method for plant sterol, it comprises:

(a) in non-human being or one or more part, improve or produce one or more following protein, described protein has Table II the 3rd row, 112-124 is capable and/or 483-491 capable shown in activity of proteins, for example have Table I the 5th or 7 row, 112-124 is capable and/or 483-491 capable shown in the sequence of the coded polypeptide of nucleic acid molecule; With

(b) allowing to produce fine chemicals, for example make biological growth under the condition of plant sterol, particularly Sitosterolum and/or campesterol.

[0016.1.12.12] therefore, term " fine chemicals " refers to " Sitosterolum " or its homologue relevant with Table I-IV 112-117 and/or the capable listed full sequence of 483-485 in one embodiment, and refers to " campesterol " or its homologue relevant with Table I-IV 118-124 and/or the capable listed full sequence of 486-491 in one embodiment.

Therefore, term " fine chemicals " refers to and Table I-IV the 113rd or 120 row and relevant " Sitosterolum " and " campesterol " of the 120th listed full sequence of row in one embodiment; Term " fine chemicals " refers to and Table I-IV the 114th or relevant " Sitosterolum " and " campesterol " of the 122 listed full sequences of row in one embodiment; In one embodiment, term " fine chemicals " refers to and Table I-IV the 115th or relevant " Sitosterolum " and " campesterol " of the 124 listed full sequences of row; Term " fine chemicals " refers to and Table I-IV the 484th or relevant " Sitosterolum " and " campesterol " of the 486 listed full sequences of row in one embodiment.

Therefore, term " fine chemicals " can refer to " Sitosterolum " and " campesterol " according to environment and context.For the meaning of illustrating term " fine chemicals " is meant " Sitosterolum " and " campesterol ", also can use term " each fine chemicals ".

[0017.0.0.12] and [0018.0.0.12] sees top paragraph [0017.0.0.0] and [0018.0.0.0] for disclosing of [0017.0.0.12] and [0018.0.0.12] these paragraphs

The method that [0019.0.12.12] produces fine chemicals advantageously causes the generation of fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare fine chemicals production high at least 10% with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as table Table II the 3rd row, protein active shown in the capable and/or 483-491 of 112-124 is capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in the capable and/or 483-491 of 112-124 was capable.

[0020.0.12.12] is surprisingly found out that, the Sitosterolum content that yeast saccharomyces cerevisiae protein YER156C, YKR057W and/or YOR084W and/or e. coli k12 protein b0019, b0421, b2699, b0050, b0161 and/or b4129 transgene expression in Arabidopis thaliana are given the plant that transforms increases.

Be surprisingly found out that the campesterol content that yeast saccharomyces cerevisiae protein YER156C, YER173W, YKR057W, YOR044W and/or YPR172W and/or e. coli k12 protein b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or b2822 transgene expression in Arabidopis thaliana are given the plant that transforms increases.

Therefore, be surprisingly found out that Sitosterolum and/or campesterol (or each fine chemicals) content that yeast saccharomyces cerevisiae protein YER156C transgene expression in Arabidopis thaliana is given the plant that transforms increase.Therefore, in one embodiment, described protein or its homologue are used to produce Sitosterolum; In one embodiment, described protein or its homologue are used to produce campesterol; In one embodiment, described protein or its homologue are used to produce Sitosterolum and campesterol.

Therefore, be surprisingly found out that Sitosterolum and/or campesterol (or each fine chemicals) content that yeast saccharomyces cerevisiae protein YKR057W transgene expression in Arabidopis thaliana is given the plant that transforms increase.Therefore, in one embodiment, described protein or its homologue are used to produce Sitosterolum; In one embodiment, described protein or its homologue are used to produce campesterol; In one embodiment, described protein or its homologue are used to produce Sitosterolum and campesterol.

Therefore, be surprisingly found out that Sitosterolum and/or campesterol (or each fine chemicals) content that e. coli k12 protein b2699 transgene expression in Arabidopis thaliana is given the plant that transforms increase.Therefore, in one embodiment, described protein or its homologue are used to produce Sitosterolum; In one embodiment, described protein or its homologue are used to produce campesterol; In one embodiment, described protein or its homologue are used to produce Sitosterolum and campesterol.

Therefore, be surprisingly found out that Sitosterolum and/or campesterol (or each fine chemicals) content that e. coli k12 protein b0161 transgene expression in Arabidopis thaliana is given the plant that transforms increase.Therefore, in one embodiment, described protein or its homologue are used to produce Sitosterolum; In one embodiment, described protein or its homologue are used to produce campesterol; In one embodiment, described protein or its homologue are used to produce Sitosterolum and campesterol.

Above [0021.0.0.12] sees for disclosing of this paragraph [0021.0.0.0]

The sequence of [0022.0.12.12] yeast saccharomyces cerevisiae YER156C has been published in Dietrich, wait (Nature387 (6632 supplementary issue), 78-81 (1997)), and its activity is not characterized still.As if it have the activity that is similar to Arabidopis thaliana putative protein matter F2K15.180.Therefore, in one embodiment, the inventive method comprises the purposes with yeast saccharomyces cerevisiae YER156C or the active protein of Arabidopis thaliana putative protein matter F2K15.180 or its homologue as shown here, as shown here its is used for producing each fine chemicals in biological or its part, it is plant sterol, for example Sitosterolum and/or campesterol and/or conjugate preferably dissociate or combining form.In another embodiment, YER156C protein expression another expression of gene in the plant sterol biosynthetic pathway increases, and preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity for example YER156C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YER156C activity of proteins of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YER173W has been published in (Nature 387 (6632 supplementary issue) such as Dietrich, 78-81,1997) and Goffeau etc. (Science 274 (5287), 546-547,1996), and its activity to be defined as be " participate in dna damage and reduction division pachytene stage check position activatory check position protein ".Therefore, in one embodiment, the inventive method comprises the yeast saccharomyces cerevisiae YER173W activity as shown here or the purposes of its homologue, as shown here, it is used for producing plant sterol in biological or its part, be campesterol and/or conjugate, particularly increase campesterol and/or conjugate, the preferred quantity of free or combining form campesterol.In another embodiment, the YER173W protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, the YER173W activity of proteins is enhanced or produces, and for example is enhanced or produces from the YER173W protein of yeast saccharomyces cerevisiae or the activity of its homologue

The sequence of yeast saccharomyces cerevisiae YKR057W has been published in Dujon etc., Nature 369 (6479), 371-378,1994 and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be to participate in that rrna is biological to be taken place and the ribosomal protein that is similar to the S21 ribosomal protein of translation.Therefore, in one embodiment, the inventive method comprises the biological protein that takes place and translate of participation rrna as shown here, particularly the protein of ribosomal protein superfamily preferably has the purposes of the active protein of S21 ribosomal protein or its homologue, and is as shown here, it is used for producing each fine chemicals in biological or its part, be plant sterol, for example Sitosterolum and/or campesterol and/or conjugate preferably dissociate or combining form.In another embodiment, the YKR057W protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, the described activity for example protein or preferably have of yeast ribosomal protein superfamily participates in that rrna is biological to be taken place and translate active activity of proteins to be enhanced or to produce, for example from the protein of the yeast ribosomal protein superfamily of yeast saccharomyces cerevisiae or preferably have and participate in the biological activity that takes place and translate active protein or its homologue of rrna and be enhanced or produce.

The sequence of yeast saccharomyces cerevisiae YOR044W has been published in Dujon etc., Nature 387 (6632 supplementary issue), and 98-102 (1997), and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises as shown here the have possible membranin YOR044w of the active protein of yeast saccharomyces cerevisiae YOR044w or yeast saccharomyces cerevisiae or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be plant sterol, for example campesterol and/or conjugate preferably dissociate or combining form.In another embodiment, the YOR044W protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity for example YOR044W activity of proteins is enhanced or produces, and for example is enhanced or produces from the YOR044W protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YOR084W has been published in Dujon, Nature 387 (6632 supplementary issue), 98-102,1997, and Goffeau, Science 274 (5287), 546-547,1996, and its activity be defined as be the supposition peroxysome matrix lipase.Therefore, in one embodiment, the inventive method comprises the peroxysome matrix lipase protein of supposition as shown here or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be plant sterol, for example Sitosterolum and/or conjugate preferably dissociate or combining form.In another embodiment, the YOR084W protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, the peroxysome matrix lipase activity of proteins that described activity is for example supposed is enhanced or produces, and for example is enhanced or produces from the peroxysome matrix lipase protein of the supposition of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YPR172W has been published in Bussey etc., Nature 387 (6632 supplementary issue), and 103-105 (1997), and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes with the active protein of yeast saccharomyces cerevisiae YPR172W or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be plant sterol, for example campesterol and/or conjugate preferably dissociate or combining form.In another embodiment, the YPR172W protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity for example YPR172W activity of proteins is enhanced or produces, and for example is enhanced or produces from the YPR172W protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0019 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity is defined as transporting the protein of small molecules, preferred cationic, i.e. Na+/H+ antiport albumen.In a more preferred embodiment, protein have Na+/H+ antiport protein-active, to coerce, particularly high salt and pH reactive activity.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary translocator, particularly Na+/H+-exchanger nhaA superfamily preferably has the protein of Na+/H+ antiport protein-active or the purposes of its homologue, and is as shown here, it is used for producing each fine chemicals in biological or its part, be plant sterol, for example Sitosterolum and/or conjugate preferably dissociate or combining form.In another embodiment, the b0019 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity for example Na+/H+-exchanger nhaA superfamily, preferably have Na+/H+ antiport protein-active the activity of Escherichia coli protein be enhanced or produce, for example from colibacillary translocator, particularly Na+/H+-exchanger nhaA superfamily, preferably have Na+/H+ antiport protein-active Escherichia coli protein or the activity of its homologue be enhanced or produce.

The sequence of e. coli k12 b0421 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be geranyltranstransferase (=method diphosphate synthase).Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary geranyltranstransferase (=method diphosphate synthase), the particularly purposes of dimethylallyltrans-transferase superfamily, geranyltranstransferase or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be plant sterol, for example Sitosterolum and/or conjugate preferably dissociate or combining form.In another embodiment, the b0421 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity for example geranyltranstransferase (=method diphosphate synthase), the particularly activity of dimethylallyltrans-transferase superfamily, geranyltranstransferase is enhanced or produces, and for example is enhanced or produces from colibacillary geranyltranstransferase (=method diphosphate synthase), the particularly activity of dimethylallyltrans-transferase superfamily, geranyltranstransferase or its homologue.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the DNA chain exchange with proteolytic enzyme and nuclease and the recombinant protein of recombinant protein recA superfamily.Therefore, in one embodiment, the inventive method comprises as shown here recombinating and the DNA repairing activity from the colibacillary DNA of having, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, the purposes of recombinant protein recA superfamily or its homologue particularly, as shown here, it is used for producing each fine chemicals in biological or its part, it is plant sterol, particularly increase Sitosterolum and/or campesterol and/or conjugate, the quantity of preferably free or combining form Sitosterolum and/or campesterol and/or conjugate.In another embodiment, the b2699 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity, for example proteolytic enzyme and nuclease, the exchange of DNA chain and recombinant protein, the particularly activity of recombinant protein recA superfamily that particularly have proteolytic enzyme and a nuclease are enhanced or produce, for example from colibacillary proteolytic enzyme and nuclease, particularly have the DNA chain exchange of proteolytic enzyme and nuclease and the activity of recombinant protein, particularly recombinant protein recA superfamily or its homologue and be enhanced or produce.

The sequence of e. coli k12 b3256 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be acetyl CoA carboxylase, biotin carboxylase enzyme subunit.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary acetyl CoA carboxylase, particularly biotin carboxylase superfamily or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be plant sterol, particularly increase campesterol and/or conjugate, the quantity of preferably free or combining form campesterol and/or conjugate.In another embodiment, the b3256 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity, acetyl CoA carboxylase activity for example, particularly the activity of biotin carboxylase superfamily is enhanced or produces, for example from colibacillary acetyl CoA carboxylase activity, particularly the activity of biotin carboxylase superfamily or its homologue is enhanced or produces.

The sequence of e. coli k12 b0050 (accession number NP_414592) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the conservative protein matter of potential participation protein-protein interaction.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary described protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, it is plant sterol, for example Sitosterolum and/or conjugate preferably dissociate or combining form.In another embodiment, the b0050 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, described activity of proteins is enhanced or produces, and for example the activity from colibacillary described protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b0161 (accession number NP_414703) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary pericentral siphon serine protease or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, it is plant sterol, for example Sitosterolum and/or campesterol and/or conjugate preferably dissociate or combining form.In another embodiment, the b0161 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, the activity of pericentral siphon serine protease is enhanced or produces, and for example the activity from colibacillary pericentral siphon serine protease or its homologue is enhanced or produces.

The sequence of e. coli k12 b4129 (accession number NP_418553) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Methionin tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary Methionin tRNA synthetase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, it is plant sterol, for example Sitosterolum and/or conjugate preferably dissociate or combining form.In another embodiment, the b4129 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, the activity of Methionin tRNA synthetase protein is enhanced or produces, and for example the activity from colibacillary Methionin tRNA synthetase protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b0464 (accession number NP_414997) has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the transcription repressor (TetR/AcrR family) of pump " multiple medicines outflow ".Therefore, in one embodiment, the inventive method comprises the purposes from intestinal bacteria " transcription repressor (TetR/AcrR family) of multiple medicines outflow pump " or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, it is plant sterol, particularly increase campesterol and/or conjugate, the quantity of preferably free or combining form campesterol and/or conjugate.In another embodiment, the b0464 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, the activity of " transcription repressor (TetR/AcrR family) of multiple medicines outflow pump " is enhanced or produces, and for example is enhanced or produces from colibacillary " transcription repressor (TetR/AcrR family) of multiple medicines outflow pump " or the activity of its homologue.

The sequence of e. coli k12 b1896 (accession number NP_416410) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be trehalose-6-phosphate synthase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary trehalose-6-phosphate synthase or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, it is plant sterol, particularly increase campesterol and/or conjugate, the quantity of preferably free or combining form campesterol and/or conjugate.In another embodiment, the b1896 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, the activity of trehalose-6-phosphate synthase is enhanced or produces, and for example the activity from colibacillary trehalose-6-phosphate synthase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2341 (accession number NP_416410) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity be defined as be " bi-functional anaerobic Fatty Acid Oxidation complex body protein: alkene acyl-CoA hydratase/epimerase/isomerase (N-end); 3-hydroxyl acyl CoA desaturase (C-end) ".Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary " bi-functional anaerobic Fatty Acid Oxidation complex body protein: alkene acyl-CoA hydratase/epimerase/isomerase (N-end); 3-hydroxyl acyl CoA desaturase (C-end) " or the purposes of its homologue; it is used for producing fine chemicals in biological or its part as described; be plant sterol, particularly increases campesterol and/or conjugate, the preferred quantity of free or combining form campesterol and/or conjugate.In another embodiment, the b2341 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, " bi-functional anaerobic Fatty Acid Oxidation complex body protein: alkene acyl-CoA hydratase/epimerase/isomerase (N-end); 3-hydroxyl acyl CoA desaturase (C-end) " activity be enhanced or produce, for example from colibacillary " bi-functional anaerobic Fatty Acid Oxidation complex body protein: alkene acyl-CoA hydratase/epimerase/isomerase (N-end); 3-hydroxyl acyl CoA desaturase (C-end) " or the activity of its homologue be enhanced or produce.

The sequence of e. coli k12 b2478 (accession number NP_416973) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be " dihydrodipicolinate synthase ".Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary dihydrodipicolinate synthase or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, it is plant sterol, particularly increase campesterol and/or conjugate, the quantity of preferably free or combining form campesterol and/or conjugate.In another embodiment, the b2478 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, dihydrodipicolinate synthase's activity is enhanced or produces, and for example the activity from colibacillary dihydrodipicolinate synthase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2822 (accession number NP_417299) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease " that its activity is defined as.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease " protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, i.e. plant sterol particularly increases campesterol and/or conjugate, the preferred quantity of free or combining form campesterol and/or conjugate.In another embodiment, the b2822 protein expression is increased with another expression of gene of plant sterol biosynthetic pathway, preferably participates in being increased from the proteinic expression of gene of middle product such as squalene and squalene epoxide or cycloartenol generation plant sterol with coding.In one embodiment, in the methods of the invention, " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease " activity of proteins is enhanced or produces, for example be enhanced or produce from colibacillary " dna helicase, ATP dependency dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease " or the activity of its homologue.

The homologous compound (=homologue) of [0023.0.12.12] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, be respectively shown in Table II the 3rd row, the 112nd, 113 or 114 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred Sitosterolum content increases.In one embodiment, homologue is the homologue that has sequence shown in Table I or II the 7th row, the 112nd, 113 or 114 row respectively.In one embodiment, be respectively shown in Table II the 3rd row, the 112nd, 113 or 114 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, be respectively the homologue of polypeptide shown in Table II the 3rd row, the 112nd, 113 or 114 row from Ascomycota.In one embodiment, be respectively the homologue of polypeptide shown in Table II the 3rd row, the 112nd, 113 or 114 row from yeast.In one embodiment, be respectively the homologue of polypeptide shown in Table II the 3rd row, the 112nd, 113 or 114 row from the yeast guiding principle.In one embodiment, the homologue that is respectively polypeptide shown in Table II the 3rd row, the 112nd, 113 or 114 row is the homologue from Saccharomycetes.In one embodiment, the homologue that is respectively polypeptide shown in Table II the 3rd row, the 112nd, 113 or 114 row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue that is respectively polypeptide shown in Table II the 3rd row, the 112nd, 113 or 114 row is to have same or similar activity and from the homologue of yeast belong.

In one embodiment, Table II the 3rd row, 118-122 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred plant sterol, preferred campesterol content increase.In one embodiment, homologue be have Table I or II the 7th row, 118-122 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, 118-122 capable shown in the polypeptide any one homologue from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd row, 118-122 capable shown in the homologue of polypeptide from Ascomycota.In one embodiment, Table II the 3rd row, 118-122 capable shown in the homologue of polypeptide from yeast.In one embodiment, Table II the 3rd row, 118-122 capable shown in the homologue of polypeptide from the yeast guiding principle.In one embodiment, Table II the 3rd row, 118-122 capable shown in the homologue of polypeptide be homologue from Saccharomycetes.In one embodiment, Table II the 3rd row, 118-122 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, 118-122 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of yeast belong.

In one embodiment, Table II the 3rd row, the 115th, 116 or 117 and 483-485 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred plant sterol, more preferably Sitosterolum content increase.In one embodiment, homologue be have the Table I of being respectively or II the 7th row, the 115th, 116 or 117 and 483-485 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, the 115th, 116 or 117 and 483-485 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, the 115th, 116 or 117 and 483-485 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, the 115th, 116 or 117 and 483-485 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, the 115th, 116 or 117 and 483-485 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, the 115th, 116 or 117 and 483-485 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, the 115th, 116 or 117 and 483-485 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, the 123rd or 124 and 486-491 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred plant sterol, more preferably campesterol content increase.In one embodiment, homologue be have the Table I of being respectively or II the 7th row, the 123rd or 124 and 486-491 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, the 123rd or 124 and 486-491 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, the 123rd or 124 and 486-491 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, the 123rd or 124 and 486-491 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, the 123rd or 124 and 486-491 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, the 123rd or 124 and 486-491 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, the 123rd or 124 and 486-491 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

[0023.1.12.12] as Table II the 3rd row, 112-124 is capable and/or the homologue of the polypeptide of 483-491 shown in capable can be by Table I the 7th row, 112-124 is capable and/or 483-491 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 112-124 is capable and/or 483-491 capable shown in polypeptide.As Table II the 3rd row, 112-124 is capable and/or 483-491 capable shown in the homologue of polypeptide can be by Table I the 7th row, 112-124 is capable and/or 483-491 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 112-124 is capable and/or 483-491 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, 112-117 and 483-485 are capable can be by be respectively Table I the 7th row, 112-117 and 483-485 capable shown in the polypeptide of nucleic acid molecule encoding, perhaps can be have the Sitosterolum content of increasing and/or quantity is active, Table II the 7th row, 112-117 and 483-485 capable shown in polypeptide.The homologue of polypeptide shown in Table II the 3rd row, 112-117 and 483-485 are capable can be by Table I the 7th row, 112-117 and 483-485 capable shown in the polypeptide of nucleic acid molecule encoding, perhaps can be have the Sitosterolum content of increasing and/or quantity is active, Table II the 7th row, 112-117 and 483-485 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, 118-124 and 486-491 are capable can be by be respectively Table I the 7th row, 118-124 and 486-491 capable shown in the polypeptide of nucleic acid molecule encoding, perhaps can be have the campesterol content of increasing and/or quantity is active, Table II the 7th row, 118-124 and 486-491 capable shown in polypeptide.The homologue of polypeptide shown in Table II the 3rd row, 118-124 and 486-491 are capable can be by Table I the 7th row, 118-124 and 486-491 capable shown in the polypeptide of nucleic acid molecule encoding, perhaps can be have the campesterol content of increasing and/or quantity is active, Table II the 7th row, 118-124 and 486-491 capable shown in polypeptide.

Above [0024.0.0.12] sees for disclosing of this paragraph [0024.0.0.0]

[0025.0.12.12] is according to the present invention, the plant sterol level increases in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, particularly Sitosterolum and/or campesterol level increase, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example be respectively Table II the 3rd row, 112-117 is capable and/or 483-485 is capable and/or 118-124 is capable and/or 486-491 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, 112-117 is capable and/or 483-485 is capable and/or 118-124 is capable and/or 486-491 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have the Table II of being respectively the 3rd row, 112-117 is capable and/or 483-485 is capable and/or 118-124 is capable and/or 486-491 capable shown in any one proteinic biology or the enzyme activity, if promptly with Table II the 3rd row, the 112nd, 113,114,118,119,120, any one yeast saccharomyces cerevisiae protein shown in 121 or 122 row is compared and/or is listed as with Table II the 3rd, the 115th, 116,117,123 or 124 or 483-491 capable shown in any one e. coli k12 protein compare, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as each fine chemicals of increase in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

Above [0025.1.0.12] and [0025.2.0.12] sees for disclosing of [0025.1.0.12] and [0025.2.0.12] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.12] to [0033.0.0.12] saw for disclosing of [0026.0.0.12] to [0033.0.0.12] these paragraphs

[0034.0.12.12] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, it has the Table II of being respectively the 3rd row, 112-117 is capable and/or 483-485 is capable and/or 118-124 is capable and/or 486-491 capable shown in protein, or by being respectively Table I the 5th row, 112-117 is capable and/or 483-485 is capable and/or 118-124 is capable and/or 486-491 capable shown in the protein of nucleic acid molecule encoding, or its homologue (as is respectively Table I the 7th row, 112-117 is capable and/or 483-485 is capable and/or 118-124 is capable and/or 486-491 capable shown in homologue) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows each fine chemicals quantity that increases.

Paragraph [0035.0.0.0] was to [0039.0.0.0] above [0035.0.0.12] to [0038.0.0.12] and [0039.0.5.12] saw for disclosing of [0035.0.0.12] to [0038.0.0.12] and [0039.0.5.12] these paragraphs

Paragraph [0035.0.0.0] and [0044.0.0.0] above [0040.0.0.12] to [0044.0.0.12] sees for disclosing of [0040.0.0.12] to [0044.0.0.12] these paragraphs

[0045.0.12.12.] in one embodiment, at yeast saccharomyces cerevisiae protein YER156C or its homologue, for example Arabidopis thaliana putative protein matter F2K15.180 is (as Table I the 5th or 7 row, shown in the 114th or 122 row) activity situation about being enhanced under, give each fine chemicals, preferred Sitosterolum be increased in 18% and 39% or more between, perhaps give each fine chemicals, preferred campesterol be increased in 17% and 95% or more between, perhaps give each fine chemicals, preferred Sitosterolum and campesterol are increased in 17% and 95%, preferably 18% and 39% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 121st row), give each fine chemicals, preferred campesterol be increased in 23% and 51% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YKR057W or its homologue, for example participate in the biological ribosomal protein that is similar to the S21 ribosomal protein that takes place and translate of rrna (as Table I the 5th or 7 row, shown in the 113rd or 120 row) activity situation about being enhanced under, give each fine chemicals, preferred Sitosterolum be increased in 14% and 32% or more between, perhaps give each fine chemicals, preferred campesterol be increased in 20% and 39% or more between, perhaps give each fine chemicals, preferred Sitosterolum and campesterol are increased in 14% and 39%, preferred 20% and 32% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR044W or its homologue, under the activity situation about being enhanced of for example possible membranin YOR044w (shown in Table I the 5th or 7 row, the 119th row), give each fine chemicals, preferred campesterol be increased in 28% and 57% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR084W or its homologue, for example under the activity situation about being enhanced of Jia Ding peroxysome matrix lipase (shown in Table I the 5th or 7 row, the 112nd row), give each fine chemicals, preferred Sitosterolum be increased in 100% and 259% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPR172W or its homologue, for example under the activity situation about being enhanced of YPR172W protein (shown in Table I the 5th or 7 row, the 118th row), give each fine chemicals, preferred campesterol be increased in 17% and 62% or more between.

In one embodiment, at e. coli k12 protein b0019 or its homologue, the protein that for example has Na+/H+ antiport protein-active, particularly under the activity situation about being enhanced of Na+/H+-exchanger nhaA superfamily (shown in Table I the 5th or 7 row, the 117th row), give each fine chemicals, preferred Sitosterolum be increased in 20% and 47% or more between.

In one embodiment, at e. coli k12 protein b0421 or its homologue, for example has the active protein of geranyltranstransferase (=method diphosphate synthase), dimethylallyltrans-transferase superfamily particularly, under the activity situation about being enhanced of geranyltranstransferase (shown in Table I the 5th or 7 row, the 116th row), give each fine chemicals, preferred Sitosterolum be increased in 13% and 52% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example has " exchange of DNA chain and recombinant protein " active protein (as Table I the 5th or 7 row with proteolytic enzyme and nuclease, shown in the 115th or 124 row) activity situation about being enhanced under, give each fine chemicals, preferred Sitosterolum be increased in 14% and 35% or more between, perhaps give each fine chemicals, preferred campesterol be increased in 17% and 62% or more between, perhaps give each fine chemicals, preferred Sitosterolum and campesterol are increased in 14% and 62%, preferred 17% and 35% or more between.

In one embodiment, at e. coli k12 protein b3256 or its homologue, for example has acetyl CoA carboxylase, the active protein of biotin carboxylase enzyme subunit, particularly under the activity situation about being enhanced of biotin carboxylase superfamily (shown in Table I the 5th or 7 row, the 123rd row), give each fine chemicals, preferred campesterol be increased in 20% and 22% or more between.

At e. coli k12 protein b0050 or its homologue, under the activity situation about being enhanced of the conservative protein matter of for example potential participation protein-protein interaction (shown in Table II the 5th or 7 row, the 483rd row), preferably, give in one embodiment fine chemicals, preferred Sitosterolum be increased in 15% and 24% or more between.

In one embodiment, at e. coli k12 protein b0161 or its homologue, for example under the activity situation about being enhanced of pericentral siphon serine protease (shown in Table II the 5th or 7 row, the 484th or 486 row), give each fine chemicals, preferred Sitosterolum be increased in 12% and 26% or more between, perhaps give each fine chemicals, preferred campesterol be increased in 17% and 42% or more between, perhaps give each fine chemicals, preferred Sitosterolum and campesterol be increased in 12% and 42%, preferred 17% and 26% or more between.

At e. coli k12 protein b4129 or its homologue, for example under the activity situation about being enhanced of Methionin tRNA synthetic enzyme (shown in Table II the 5th or 7 row, the 485th row), preferably, give in one embodiment fine chemicals, preferred Sitosterolum be increased in 21% and 23% or more between.

In one embodiment, at e. coli k12 protein b0464 or its homologue, for example under the situation that the activity of the transcription repressor (TetR/AcrR family) of multiple medicines outflow pump (shown in Table II the 5th or 7 row, the 487th row) is enhanced, preferably, give in one embodiment fine chemicals, preferred campesterol be increased in 16% and 32% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the activity situation about being enhanced of trehalose-6-phosphate synthase (shown in Table II the 5th or 7 row, the 488th row), preferably, give in one embodiment fine chemicals, preferred campesterol be increased in 18% and 59% or more between.

In one embodiment, at e. coli k12 protein b2341 or its homologue, " bi-functional anaerobic Fatty Acid Oxidation complex body protein: alkene acyl-CoA hydratase/epimerase/isomerase (N-end) for example; 3-hydroxyl acyl CoA desaturase (C-end) " under the activity situation about being enhanced of (shown in Table II the 5th or 7 row, the 489th row), preferably, give in one embodiment fine chemicals, preferred campesterol be increased in 18% and 48% or more between.

In one embodiment, at e. coli k12 protein b2478 or its homologue, for example under dihydrodipicolinate synthase's's (shown in Table II the 5th or 7 row, the 490th row) the activity situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred campesterol be increased in 17% and 37% or more between.

At e. coli k12 protein b2822 or its homologue, under the activity situation about being enhanced of for example " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease " (shown in Table II the 5th or 7 row, the 491st row), preferably, give in one embodiment fine chemicals, preferred campesterol be increased in 21% and 32% or more between.

[0046.0.12.12] in one embodiment, yeast saccharomyces cerevisiae protein YER156C or its homologue, for example the activity of Arabidopis thaliana putative protein matter F2K15.180 (shown in Table I the 5th or 7 row, the 114th or 122 row) is given each fine chemicals and other is had the active compound of plant sterol or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YER173W or its homologue, the activity that for example participates in dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 121st row) are given each fine chemicals and other are had the active compound of plant sterol or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YKR057W or its homologue for example participate in that rrna is biological to be taken place and the activity of the ribosomal protein that is similar to the S21 ribosomal protein of translation (shown in Table I the 5th or 7 row, the 113rd or 120 row) is given each fine chemicals and other had the active compound of plant sterol or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YOR044W or its homologue, the activity of for example possible membranin YOR044w (shown in Table I the 5th or 7 row, the 119th row) are given each fine chemicals and other are had the active compound of plant sterol or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YOR084W or its homologue, for example the activity of Jia Ding peroxysome matrix lipase (shown in Table I the 5th or 7 row, the 112nd row) is given each fine chemicals and other is had the active compound of plant sterol or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YPR172W or its homologue, for example the activity of YPR172W protein (shown in Table I the 5th or 7 row, the 118th row) is given each fine chemicals and other is had the active compound of plant sterol or its precursor increases.

In one embodiment, e. coli k12 protein b0019 or its homologue for example have the protein of Na+/H+ antiport protein-active.Particularly the activity of Na+/H+-exchanger nhaA superfamily (shown in Table I the 5th or 7 row, the 117th row) is given each fine chemicals and other is had the active compound of plant sterol or its precursor increases.

In one embodiment, e. coli k12 protein b0421 or its homologue, for example has the active protein of geranyltranstransferase (=method diphosphate synthase), dimethylallyltrans-transferase superfamily particularly, the activity of geranyltranstransferase (shown in Table I the 5th or 7 row, the 116th row) are given each fine chemicals and other are had the active compound of plant sterol or its precursor increases.

In one embodiment, e. coli k12 protein b2699 or its homologue, the activity that for example has " DNA chain exchange and recombinant protein with proteolytic enzyme and nuclease " active protein (shown in Table I the 5th or 7 row, the 115th or 124 row) are given each fine chemicals and other are had the active compound of plant sterol or its precursor increases.

In one embodiment, e. coli k12 protein b3256 or its homologue, the activity that for example has an acetyl CoA carboxylase, the active protein of biotin carboxylase enzyme subunit, particularly biotin carboxylase superfamily (shown in Table I the 5th or 7 row, the 123rd row) is given each fine chemicals and other is had the active compound of plant sterol or its precursor increases.

In one embodiment, at e. coli k12 protein b0050 or its homologue, under the situation that the activity of the conservative protein matter of for example potential participation protein-protein interaction is enhanced, preferably give fine chemicals and other have the active compound of plant sterol or its precursor increases.

At e. coli k12 protein b0161 or its homologue, for example under the situation that the activity of pericentral siphon serine protease is enhanced, preferably, giving one or more fine chemicals in one embodiment increases, and preferably gives one or more fine chemicals and other has the active compound of plant sterol or its precursor increases.

In one embodiment, at e. coli k12 protein b4129 or its homologue, for example under the situation that the activity of Methionin tRNA synthetic enzyme is enhanced, preferably, giving fine chemicals in one embodiment increases, and preferably gives fine chemicals and other has the active compound of plant sterol or its precursor increases.

In one embodiment, at e. coli k12 protein b0464 or its homologue, for example under the situation that the activity of the transcription repressor (TetR/AcrR family) of multiple medicines outflow pump is enhanced, preferably, giving fine chemicals in one embodiment increases, and preferably gives fine chemicals and other has the active compound of plant sterol or its precursor increases.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that the activity of trehalose-6-phosphate synthase is enhanced, preferably, giving fine chemicals in one embodiment increases, and preferably gives fine chemicals and other has the active compound of plant sterol or its precursor increases.

In one embodiment, at e. coli k12 protein b2341 or its homologue, " bi-functional anaerobic Fatty Acid Oxidation complex body protein: alkene acyl-CoA hydratase/epimerase/isomerase (N-end) for example; 3-hydroxyl acyl CoA desaturase (C-end) " activity situation about being enhanced under, preferably, giving fine chemicals in one embodiment increases, and preferably gives fine chemicals and other has the active compound of plant sterol or its precursor increases.

In one embodiment, at e. coli k12 protein b2478 or its homologue, for example under the situation that dihydrodipicolinate synthase's activity is enhanced, preferably, giving fine chemicals in one embodiment increases, and preferably gives fine chemicals and other has the active compound of plant sterol or its precursor increases.

In one embodiment, at e. coli k12 protein b2822 or its homologue, for example under the situation that the activity of " dna helicase; ATP dependency dsDNA/ssDNA exonuclease V subunit; ssDNA endonuclease " is enhanced, preferably, giving fine chemicals in one embodiment increases, and preferably gives fine chemicals and other has the active compound of plant sterol or its precursor increases.

Paragraph [0047.0.0.0] and [0048.0.0.0] above [0047.0.0.12] to [0048.0.0.12] sees for disclosing of [0047.0.0.12] and [0048.0.0.12] these paragraphs

[0049.0.7.7] has to give and improves Sitosterolum quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, consensus sequence shown in 112-117 and 483-485 are capable and/or as Table IV the 5th or 7 row, the sequence of consensus sequence shown in 112-117 and 483-485 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 112-117 and 483-485 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 112-117 and 483-485 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the Sitosterolum level of giving described in the literary composition increases.

Have to give and improve campesterol quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, consensus sequence shown in 118-124 and 486-491 are capable and/or as Table IV the 5th or 7 row, the sequence of consensus sequence shown in 118-124 and 486-491 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 118-124 and 486-491 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 118-124 and 486-491 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the campesterol level of giving described in the literary composition increases.

[0050.0.12.12] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example the sylvite of plant sterol or sodium salt or its ester.

[0051.0.5.12] and [0052.0.0.12] sees top paragraph [0051.0.0.0] and [0052.0.0.0] for disclosing of [0051.0.5.12] and [0052.0.0.12] these paragraphs

[0053.0.12.12] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein give the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide) express and increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention (for example have as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention or (for example have as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by the coded protein of nucleic acid molecule of the present invention, polypeptide perhaps of the present invention (for example has as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide) express to improve;

(e) by add the activity that one or more external source inducible factors come stimulating protein in biological or its part, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide) express to improve;

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide);

(h) by add positive Expression element or remove negative Expression element improve code book invention polypeptide (for example have as Table II the 3rd row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.12.12] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having as being respectively Table II the 5th row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in protein or its homologue (for example be respectively Table II the 5th row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in polypeptide) give each fine chemicals after active expression of polypeptides or the activity and increase.

Paragraph [0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.12] to [0067.0.0.12] saw for disclosing of [0055.0.0.12] to [0067.0.0.12] these paragraphs

[0068.0.12.12] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of plant sterol.

Above [0069.0.0.12] sees for disclosing of this paragraph [0069.0.0.0]

[0070.0.12.12] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, YER156C perhaps encodes, YKR057W, YOR084W, b0019, b0421, b2699, b0050, b0161 and/or b4129 protein or YER156C, YER173W, YKR057W, YOR044W, YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, a gene in b2478 and/or the b2822 protein or a plurality of gene import biology separately or with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example contain the favourable fatty acid composition of plant sterol etc.

Above [0071.0.5.12] sees for disclosing of this paragraph [0071.0.0.0]

[0072.0.12.12] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has other sterol, stanols or squalene, squalene epoxide or cycloartenol except plant sterol.

[0073.0.12.12] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or its homologue (for example Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and optional other free and/or bonded plant sterol or its conjugate.

[0074.0.12.12] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded or free and each fine chemicals of bonded, and if dissociate or/and bonded plant sterol or its conjugate as selecting to produce, reclaim and expect to separate other.

Paragraph [0075.0.0.0] was to [0077.0.0.0] above [0075.0.0.12] to [0077.0.0.12] saw for disclosing of [0075.0.0.12] to [0077.0.0.12] these paragraphs

[0078.0.12.12] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes, for example (it discloses treatment experimenter hyperlipidaemia to reduce the method for low-density lipoprotein (" LDL ") level according to US 20040101829.), (it discloses the preparation of compositions that comprises sterol ester to US 20040047971, it is characterized in that the direct transesterify preparation by sterol and triacylglycerol.), (it has described the composition based on plant sterol that is used to prevent and treats cardiovascular disorder and other disease to US 5,965,449.), (it has described the pharmaceutical composition that comprises Sitosterolum that is used for the treatment of the diabetic male sexual disorder to US 5,523,087.), (it discloses and has suppressed fat and the cholesterol composition from intestinal absorption US 5,747,464, and said composition comprises the β-Gu Zaichun of irreversible fixation to pectin.), (it has described the vitamin replenisher that contains the phytosterol fatty acid ester to US 4,588,717.), (it has taught the purposes that a small amount of sterol, its fatty acid ester and glucosides are used for the treatment of tumour to US 5,270,041.), US 6,087,353 (it comprises and makes the method for compositions be suitable for being mixed into food, beverage, medicine, class medicament nutritious prod etc., it comprise with suitable aliphatic acid and plant sterol condensation with form plant sterol ester and subsequently with the plant sterol ester hydrogenation with formation hydrogenant plant sterol ester.) in their clear in this article quoting are as a reference disclosed.

In order to extract plant sterol, can water and organic solvent (hexane and acetone) processing fermented liquid, tunning, plant or plant product.By remove sterol and the calcium chloride complexing in the methyl alcohol, centrifugation sterol mixture in solvent, the extract, the thermal degradation mixture closes removal water in water, the thick plant sterol of acquisition from organic phase.By being further purified thick plant sterol from the Virahol crystallization.According to another kind of production process, tall oil soap at first carries out fractional distillation to remove volatile compound.The resulting resistates (tall oil pitch) that comprises the esterified form sterol with alkaline purification so that discharge these sterols.After the neutralization, material is carried out two phase still-process.Then overhead product is dissolved in methyl alcohol/methylethyl ketone solvent, and by filtering, obtaining crystallization of sterols with solvent wash and drying from this solution.US 4,420, and 427 professors use and prepare sterol such as methanol solvent from the vegetables oil sludge.Alternatively, can obtain plant sterol from tall oil pitch or soap (byproduct of forest estate) as quoting in the literary composition described in the PCT/CA95/00555 as a reference.Know with this paper other method described below by those skilled in the art and can extract and crystallization.

According to US 6,087,353, in order to form plant sterol ester, selected plant sterol and aliphatic acid or its ester and volatility ethanol are mixed under reaction conditions, allow plant sterol and aliphatic acid condensation generation ester.Prepare these most preferred methods that are widely used in the ester of edible-fat and oil industry and be described in U.S. Patent No. 5,502,045 (quoting as a reference in this patent literary composition).By directly preferred catalytic esterification method such as U.S. Patent No. 5,892,068 between the free fatty acids of composition or lipid acid admixture and stanols and/or sterol, also can produce stanols and/or sterol ester with expection fatty acid composition.In addition, by also producing stanols and/or sterol ester as the enzyme esterification of being summarized among the EP 195 311 (quoting as a reference in this patent literary composition).

For these different methods of setting up gradually, product is plant sterol and/or ester and/or conjugate or composition, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

Paragraph [0079.0.0.0] was to [0084.0.0.0] above [0079.0.0.12] to [0084.0.0.12] saw for disclosing of [0079.0.0.12] to [0084.0.0.12] these paragraphs

[0085.0.12.12] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as be respectively Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as be respectively Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.12] and [0087.0.0.12] sees top paragraph [0086.0.0.0] and [0087.0.0.0] for disclosing of [0086.0.0.12] and [0087.0.0.12] these paragraphs

[0088.0.12.12] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified plant sterol content.Because the nutritive value that for example is used for the plant of raise poultry depends on above-mentioned plant sterol and as the total number of the plant sterol of feed and/or food source, so this is very important for the plant breeder.In addition, but because the stress resistance of the balanced contents inducing plant of different plant sterols, so this also is important.After YER156C, YKR057W, YOR084W, b0019, b0421, b2699, b0050, b0161 and/or b4129 and/or YER156C, YER173W, YKR057W, YOR044W, YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or b2822 protein active are enhanced or produce, perhaps after nucleic acid molecule according to the present invention or polypeptide expression are produced or are improved, the transgenic plant that produced are grown on the nutritional medium or among, otherwise it is grown in the soil, subsequently results.

[0088.1.0.12], [0089.0.0.12] and [0090.0.0.12] are for [0088.1.0.12], and disclosing of [0089.0.0.12] and [0090.0.0.12] these paragraphs seen top paragraph [0088.1.0.0], [0089.0.0.0] and [0090.0.0.0]

[0091.0.12.12] therefore, the plant component foreign matter content is low as far as possible, and preferably other foreign matter contents are also low as far as possible, and the aforementioned plant sterol that obtains is pure form as far as possible.In these are used, the content of plant component favourable for by weight less than 10%, preferred 1%, more preferably 0.1%, very particularly preferably 0.01% or lower.

Paragraph [0092.0.0.0] was to [0094.0.0.0] above [0092.0.0.12] to [0094.0.0.12] saw for disclosing of [0092.0.0.12] to [0094.0.0.12] these paragraphs

[0095.0.12.12] advantageously, the pond that increases plant sterol by method of the present invention in genetically modified organism is to separate a large amount of pure fine chemicals.

[0096.0.12.12] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals such as Sitosterolum or campesterol in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.

Above [0097.0.0.12] sees for disclosing of this paragraph [0097.0.0.0]

[0098.0.12.12] in preferred embodiments, each fine chemicals (Sitosterolum or campesterol) be produce according to the present invention and carry out isolating where necessary.The method according to this invention produce other plant sterol or conjugate or its mixture or with the mixture of other compound be favourable.

For microbial fermentation, aforementioned plant sterol (preferred Sitosterolum and/or campesterol) can be accumulated in substratum and/or the cell [0099.0.12.12].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or substratum of its growth, perhaps from biology and substratum, separate the composition that comprises each fine chemicals or plant sterol (preferably Sitosterolum and/or campesterol).These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.12.12] comprises the transgenic plant that the method according to this invention synthetic comprises plant sterol (preferred Sitosterolum and/or campesterol) and can advantageously directly put on market, and do not need institute's synthetic plant sterol (preferred Sitosterolum and/or campesterol) (institute's synthetic oils, lipid or lipid acid) is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet, each fine chemicals that the method according to this invention produces can also with the form of its oils, fat, lipid, ester class and/or as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free plant sterol from biology, advantageously from plant, separate.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part, preferred plant seed.In order to improve the efficient of extraction, vegetable material, particularly seed are cleaned, soften (temper) and shell in case of necessity and peel off is favourable.In this context, oils, fat, lipid, ester class and/or free plant sterol can need not heat cold be beaten or cold-press process obtains by being called.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.In this mode, more than 96% of compound that separable the inventive method produces.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

The sterol of plant (plant sterol) is the byproduct during the traditional plant olein is practiced.The normally thick edible oil mixture in source, it is made of in varing proportions soybean oil or other edible oil such as Semen Maydis oil, Semen Brassicae campestris, sweet oil and plam oil.Hemp and sea-buckthorn ( Rhamnoides) also be the new source of containing seed, oil and food composition.By squeezing or the crude oil that obtains of solvent extraction need also that a series for the treatment of process is desolvated to remove, Yelkin TTS, free fatty acids, chromoplastid, deodorization and take off local flavor.In one of these steps, oil vapor distillation (deodorization) and resulting distillment under the pressure that reduces are contained the plant sterol fraction.Thereby by fractionation, ethanol decomposition/transesterification, distillation and from n-heptane solution crystallization from this fraction, remove lipid acid, Yelkin TTS and other compound, and be further purified plant sterol by recrystallization by using food-grade raw material and food to make convention.Extraction and purification step are standard methods, and similar with the conventional method of using of the foodstuffs industry of producing plant sterol.Use is from the lipid acid of vegetables oil or triacylglycerol and use fat and esterification that oils industry is used usually or the standard method of transesterification can be produced plant sterol ester from sterol.

Therefore plant sterol may be positioned at cell in the microorganism, must relate to separation to biomass to their recovery.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.The mensuration of tocopherol was carried out description by Tan and Tsumura in 1989 in the pair cell, was also shown in the Biotechnology ofVitamins that Erik J.Vandamme edits, Pigments and Growth Factors, London, 1989, the 96-103 pages or leaves.Many other methods of measuring tocopherol content are well known by persons skilled in the art.

[0101.0.12.12] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology ", the 17th volume.

[0102.0.12.12] for example, plant sterol can advantageously be analyzed and detect by MS oder MSMS method by HPLC or GC separation method.By using standard method of analysis (GC, GC-MS or TLC) can clearly detect the existence of the product that contains Sitosterolum and/or campesterol to the biology analysis of recombinating, described standard method of analysis such as Christie and reference (1997 wherein, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric methods], Lipide 33:343-353) and described.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed; Also referring to Biotechnology of Vitamins, Pigments and Growth Factors, Erik J.Vandamme edits, London, 1989, p.96-103.

For the separation and the analysis of sterol, can use German standard method F III (1).This method comprises: fatty saponification, use alumina column separate unsaponified material, by preparation type TLC sterols separated fraction and by the sterol composition of GLC mensuration as the trimethyl silyl ester.

[0103.0.12.12] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) the preferred nucleic acid molecule of encoding mature form polypeptide at least, described polypeptide have as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or the sequence of 486-491 shown in capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or the sequence of 486-491 shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use have as Table III the 7th row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable or 118-124 is capable or 486-491 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.0.12.] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule IIA the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of sequence.

[00103.2.0.12.] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of sequence.

[0104.0.12.12] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of sequence.

Paragraph [0105.0.0.0] and [0107.0.0.0] above [0105.0.0.12] to [0107.0.0.12] sees for disclosing of [0105.0.0.12] to [0107.0.0.12] these paragraphs

Advantageously improved in [0108.0.12.12] method of the present invention and had Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleic acid molecule of sequence, from Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in aminoacid sequence derive or from containing Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the polypeptide deutero-nucleic acid molecule of consensus sequence, perhaps its coding has as Table II the 3rd, 5 or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the enzymic activity of polypeptide or bioactive polypeptide or for example to give each fine chemicals behind its expression or active the increasing be plant sterol, the derivative or the homologue that increase of Sitosterolum and/or campesterol particularly.

Above [0109.0.0.12] sees for disclosing of this paragraph [0109.0.0.0]

[0110.0.12.12] helps the inventive method and coding has the polypeptide of the used polypeptide active of the used or of the present invention process of the inventive method (for example as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in protein by as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the protein of nucleic acid molecule encoding or its homologue (as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in)) nucleic acid molecule can from generally open database, determine.

Above [0111.0.0.12] sees for disclosing of this paragraph [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.12.12] the inventive method is the isolated nucleic acid sequences form, its coding have as Table II the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide active polypeptide or have as Table II the 5th and 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the polypeptide of peptide sequence, and give plant sterol, preferred Sitosterolum and/or campesterol level and increase.

Paragraph [0113.0.0.0] and [0120.0.0.0] above [0113.0.0.12] to [0120.0.0.12] sees for disclosing of [0113.0.0.12] to [0120.0.0.12] these paragraphs

[0121.0.12.12] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in peptide sequence or its functional homologue as herein described the difference of one or more amino acid moleculars is arranged, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, giving the Sitosterolum level shown in the capable and/or 483-485 of 112-117 is capable after the activity of peptide sequence increases, and is perhaps improving Table II the 5th or 7 row, giving the campesterol level shown in the capable and/or 486-491 of 118-124 is capable after the activity of peptide sequence increases.

Paragraph [0122.0.0.0] and [0127.0.0.0] above [0122.0.0.12] to [0127.0.0.12] sees for disclosing of [0122.0.0.12] to [0127.0.0.12] these paragraphs

The synthetic oligonucleotide primer thing that [0128.0.12.12] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the primer of 486-491 shown in capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence or from as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence deutero-sequence produce.

[0129.0.12.12] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in consensus sequence from described comparison.

[0130.0.12.12] can use degenerated primer to have the new protein fragment of aforementioned activity (giving plant sterol, preferred Sitosterolum and/or campesterol increase as comprise this segmental protein expression or activity in raising after) by pcr amplification then.

Paragraph [0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.12] to [0138.0.0.12] saw for disclosing of [0131.0.0.12] to [0138.0.0.12] these paragraphs

[0139.0.12.12] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence hybridization, and coding express have the plant sterol of increasing, preferred Sitosterolum and/or the active peptide of campesterol.

Paragraph [0140.0.0.0] was to [0146.0.0.0] above [0140.0.0.12] to [0146.0.0.12] saw for disclosing of [0140.0.0.12] to [0146.0.0.12] these paragraphs

[0147.0.12.12] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.12.12] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in nucleotide sequence or its portion homologous at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly having after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue increases plant sterol, the activity of Sitosterolum and/or campesterol particularly.

[0149.0.12.12] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one of nucleotide sequence or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and (as gives plant sterol, particularly Sitosterolum and/or campesterol increase) and randomly as Table II the 5th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the protein of protein active.

[00149.1.12.12] randomly, in one embodiment, with Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the protein of 486-491 shown in capable is known activity or is used for these protein of note.

[0150.0.12.12] in addition, nucleic acid molecule of the present invention can only contain Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give plant sterol, particularly Sitosterolum and/or campesterol increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one of sequence sense strand, Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the right PCR of primer will produce as Table I the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in fragment or its gene product of polynucleotide sequence.

Above [0151.0.0.12] sees for disclosing of this paragraph [0151.0.0.0]

[0152.0.12.12] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising plant sterol, particularly Sitosterolum as be shown in the examples and/or campesterol level.

[0153.0.12.12] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in protein or its part have Table II the 3rd row for example as herein described, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the activity of polypeptide.

[0154.0.12.12] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.12] and [0156.0.0.12] sees top paragraph [0155.0.0.0] and [0156.0.0.0] for disclosing of [0155.0.0.12] and [0156.0.0.12] these paragraphs

[0157.0.12.12] the present invention relates in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example giving each fine chemicals increase in the biology) is for example by Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence encoding polypeptide or as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment as Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the consensus sequence of 486-491 shown in capable or as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the consensus sequence of 486-491 shown in capable or as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet, in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in nucleic acid molecule identical.

Paragraph [0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.12] to [0160.0.0.12] saw for disclosing of [0158.0.0.12] to [0160.0.0.12] these paragraphs

[0161.0.12.12] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

Above [0162.0.0.12] sees for disclosing of this paragraph [0162.0.0.0]

[0163.0.12.12] preferably, under stringent condition with Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

Above [0164.0.0.12] sees for disclosing of this paragraph [0164.0.0.0]

[0165.0.12.12] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.12] and [0167.0.0.12] sees top paragraph [0166.0.0.0] and [0167.0.0.0] for disclosing of [0166.0.0.12] and [0167.0.0.12] these paragraphs

[0168.0.12.12] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the aminoacid sequence of 486-491 shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or one of the sequence of 486-491 shown in capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable at least about 96%, 97%, 98% or 99% is identical.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the aminoacid sequence of 486-491 shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or one of the sequence of 486-491 shown in capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable at least about 96%, 97%, 98% or 99% is identical.

Paragraph [0169.0.0.0] was to [0174.0.0.0] above [0169.0.0.12] to [0174.0.0.12] saw for disclosing of [0169.0.0.12] to [0174.0.0.12] these paragraphs

[0175.0.12.12] for example, the sequence that has 80% homology at protein level and SEQ ID NO:12136 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:12136 sequence.

[0176.0.12.12] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the functional equivalent that obtains of one of polypeptide be listed as according to of the present invention as Table II the 5th or 7,112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide have essentially identical character and discern.

[0177.0.12.12] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the functional equivalent that obtains of nucleotide sequence be listed as according to of the present invention as Table II the 5th or 7,112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the polypeptide of the essentially identical character of polypeptide.

Above [0178.0.0.12] sees for disclosing of this paragraph [0178.0.0.0]

[0179.0.12.12] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in) in introduce the replacement of one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 introduces sudden change in the encoding sequence of sequence shown in capable.

Paragraph [0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.12] to [0183.0.0.12] saw for disclosing of [0180.0.0.12] to [0183.0.0.12] these paragraphs

[0184.0.12.12] is employed to have as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleotide sequence homologous compound of sequence, perhaps come Table II the 5th or 7 row freely, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the homologous compound of nucleotide sequence of sequence also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.12.12] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the arbitrary sequence of 486-491 shown in capable in other Nucleotide of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.12.12] also preferred the inventive method comprise as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence identical.

[0187.0.12.12] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the encoding sequence of aminoacid sequence identical.

[0188.0.12.12] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in and under the same conditions the polypeptide expressed activity compare and basic do not reduce.In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence or by its homologue of forming.

[0189.0.12.12] as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the homologous compound of sequence, or deutero-as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the homologue of the sequence of 486-491 shown in capable also refers to the single stranded DNA or the RNA of truncated sequence, eDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

Paragraph [0190.0.0.0] was to [0203.0.0.0] above [0190.0.0.12] to [0203.0.0.12] saw for disclosing of [0190.0.0.12] to [0203.0.0.12] these paragraphs

[0204.0.12.12] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding is being listed as Table II the 5th or 7 of mature form at least, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or polypeptide or its segmental nucleic acid molecule of 486-491 shown in capable, described nucleic acid molecule is given each fine chemicals in biological or its part, i.e. plant sterol, particularly Sitosterolum (112-117 is capable and/or 483-485 is capable) and/or campesterol (118-124 is capable and/or 486-491 is capable) quantity increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or nucleic acid molecule or its segmental nucleic acid molecule of 486-491 shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the primer of 486-491 shown in capable or primer obtain amplification from cDNA library or genomic library nucleic acid molecule, and in biological or its part, give each fine chemicals, i.e. the increase of plant sterol, particularly Sitosterolum (112-117 is capable) and/or campesterol (118-124 is capable) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in consensus sequence and in biological or its part, give each fine chemicals, the i.e. nucleic acid molecule that increases of plant sterol, particularly Sitosterolum (112-117 is capable and/or 483-485 is capable) and/or campesterol (118-124 is capable and/or 486-491 is capable) quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be the nucleic acid molecule that plant sterol, particularly Sitosterolum (112-117 is capable and/or 483-485 is capable) and/or campesterol (118-124 is capable and/or 486-491 is capable) quantity increase, described peptide coding as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the structural domain of the polypeptide of 486-491 shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7,112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the nucleic acid molecule of 486-491 shown in capable or coding (optimized encoding is mature form at least) as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table II B the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the 15nt at least of nucleic acid molecule of polypeptide, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby, the nucleic acid molecule of preferred (a) to (l) by one or more Nucleotide be different from as Table I A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable.In one embodiment, nucleic acid molecule not by Table I A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable formed.In one embodiment, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In other embodiments, nucleic acid molecule coding schedule IIA the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of sequence.Therefore, in one embodiment, with Table II A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide have the different polypeptide of one or more amino acid do not encode as Table II A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the protein of sequence.Therefore, in one embodiment, by nucleotide sequence (a)-(l) encoded protein matter not by as Table II A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in protein sequence at least 30% identical, and with Table II A the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% identical.

[0205.0.0.12] and [0206.0.0.12] sees top paragraph [0205.0.0.0] and [0206.0.0.0] for disclosing of [0205.0.0.12] and [0206.0.0.12] these paragraphs

[0207.0.12.12] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of sterol metabolism, squalene metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

Paragraph [0208.0.0.0] was to [0226.0.0.0] above [0208.0.0.12] to [0226.0.0.12] saw for disclosing of [0208.0.0.12] to [0226.0.0.12] these paragraphs

[0227.0.12.12] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the capable sequence or derivatives thereof of mentioning of 486-491, can be additionally advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one sterol (for example plant sterol precursor such as squalene epoxide) biosynthetic pathway in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.12.12] therefore cultivated to cross simultaneously in another embodiment of the present invention and expressed coding and sterol/plant sterol metabolism (particularly Sitosterolum and/or campesterol are synthetic) at least one nucleic acid of related protein or the biology of a gene.

[0229.0.12.12] can make up the sequence of other favourable nucleotide sequences of expression as other gene of coded plant sterol biosynthetic pathway with sequence that present method is used and/or aforementioned biosynthesis gene, acetyl-CoA HMGCoA for example, mevalonic acid, mevalonic acid 5 phosphoric acid, mevalonic acid 5-tetra-sodium, the isopentyl bisphosphate, 5-pyrophosphomevalonic acid, isopentyl tetra-sodium (PIP), dimethyl allene tetra-sodium (DMAPP), PIP+DMAPP, geranylpyrophosphate+IPP, farnesylpyrophosphate, 2 farnesylpyrophosphates, the enzyme that squalene (squalene synthase) and squalene epoxide produce is perhaps controlled the cycloartenol synthase of squalene epoxide cyclisation, S-adenosine-L-the methionine(Met) of methyl is shifted in catalysis from cofactor: sterol C-24 methyltransgerase (EC 2.1.1.41) (SMT1), the SMT2 of catalysis Methyl transporters reaction for the second time, catalysis C-14 position demethylation, remove the sterol C-14 demethylase of methyl and the two keys of generation.These genes can cause essential plant sterol, particularly Sitosterolum and/or the synthetic increase of campesterol.

Above [0230.0.0.12] sees for disclosing of this paragraph [0230.0.0.0]

[0231.0.12.12] is in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degrading plant sterol, particularly Sitosterolum and/or campesterol simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

Paragraph [0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.5] to [0276.0.0.5] saw for disclosing of [0232.0.0.5] to [0276.0.0.5] these paragraphs

[0277.0.12.12] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.Each fine chemicals of producing by this process can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing plant part or extract realization.

Paragraph [0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.12] to [0282.0.0.12] saw for disclosing of [0278.0.0.12] to [0282.0.0.12] these paragraphs

[0283.0.12.12] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in protein, perhaps as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the antibody of polypeptide, it can utilize polypeptide of the present invention or its fragment to produce by standard technique.Monoclonal antibody preferably.

Above [0284.0.0.12] sees for disclosing of this paragraph [0284.0.0.0]

[0285.0.12.12] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence by as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.12.12] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in consensus sequence or and in another embodiment by the active of its polypeptide of forming, the present invention relates to contain just like Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in consensus sequence or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the polypeptide of consensus sequence (each row).

Paragraph [0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.12] to [0290.0.0.12] saw for disclosing of [0287.0.0.12] to [0290.0.0.12] these paragraphs

[0291.0.12.12] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the sequence of 486-491 shown in capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence form.

Above [0292.0.0.12] sees for disclosing of this paragraph [0292.0.0.0]

[0293.0.12.12] the present invention relates to give that each fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, described polypeptide have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in other sequence of sequence phase region.In another embodiment, polypeptide of the present invention not by Table II A or IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence form.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.

[0294.0.12.12] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the polypeptide of activity of proteins, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence.

Paragraph [0295.0.0.0] was to [0297.0.0.0] above [0295.0.0.12] to [0297.0.0.12] saw for disclosing of [0295.0.0.12] to [0297.0.0.12] these paragraphs

The chemical of [0297.1.12.12] non-polypeptide of the present invention is for example not have Table II the 3rd, 5 or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the active polypeptide of polypeptide.

[0298.0.12.12] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the identical aminoacid sequence of sequence.

[0299.0.12.12] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in one of nucleotide sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with capable as Table I 112-117 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.12.12] therefore, described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence different.Therefore, this polypeptide contain with as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

Above [0301.0.0.12] sees for disclosing of this paragraph [0301.0.0.0]

The biologically-active moiety of [0302.0.12.12] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the aminoacid sequence of 486-491 shown in capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

Above [0303.0.0.12] sees for disclosing of this paragraph [0303.0.0.0]

[0304.0.12.12] operation nucleic acid molecule of the present invention may cause generation have basically as Table II the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.12], [0306.0.0.12] and [0306.1.0.12] are for [0305.0.0.12], and disclosing of [0306.0.0.12] and [0306.1.0.12] these paragraphs seen top paragraph [0305.0.0.0], [0306.0.0.0] and [0306.1.0.0]

[0307.0.0.12] and [0308.0.0.12] sees top paragraph [0307.0.0.0] and [0308.0.0.0] for disclosing of [0307.0.0.12] and [0308.0.0.12] these paragraphs

[0309.0.12.12] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the protein of 486-491 shown in capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, the polypeptide that 112-117 is capable or 483-485 is capable and/or 118-124 is capable or " the non-of the present invention polypeptide " or " other polypeptide " of 486-491 in capable is meant the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is proteinic and from the protein of identical or different biology shown in capable.In one embodiment, be not shown in " the non-polypeptide of the present invention " or " other polypeptide " that Table II the 5th or 7 is listed as, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable and do not give each fine chemicals increase in biology or its part.

Paragraph [0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.12] to [0334.0.0.12] saw for disclosing of [0310.0.0.12] to [0334.0.0.12] these paragraphs

[0335.0.12.12] confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the expression of the nucleotide sequence of 486-491 shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

Paragraph [0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.12] to [0342.0.0.12] saw for disclosing of [0336.0.0.12] to [0342.0.0.12] these paragraphs

[0343.0.12.12] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

Paragraph [0344.0.0.0] was to [0361.0.0.0] above [0344.0.0.12] to [0361.0.0.12] saw for disclosing of [0344.0.0.12] to [0361.0.0.12] these paragraphs

[0362.0.12.12] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the polypeptide of 486-491 shown in capable, and for example coding has protein such as Table II the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the polypeptide of polypeptide active) nucleic acid molecule.

Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Have as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide (for example have as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

Above [0363.0.0.12] sees for disclosing of this paragraph [0363.0.0.0]

[0364.0.12.12] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---for example naturally occurring as Table II the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide of the present invention promotor with accordingly as Table I the 3rd row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the combination of protein coding sequence---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.12] to [0373.0.0.12] for [0365.0.0.12], disclosing of these paragraphs of-[0373.0.0.12] sees that top paragraph [0365.0.0.0] is to [0373.0.0.0]

[0374.0.12.12] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the plant sterol that produces in the inventive method, particularly Sitosterolum and/or campesterol also can be from plant with free or be bonded to the isolated in form of compound or part (moiety).Plant sterol, particularly Sitosterolum that produces by this method and/or campesterol can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.12] and [0376.0.0.12] sees top paragraph [0375.0.0.0] and [0376.0.0.0] for disclosing of [0375.0.0.12] and [0376.0.0.12] these paragraphs

[0377.0.12.12] therefore the invention still further relates to the method according to this invention, separated the composition that contains plant sterol, particularly Sitosterolum and/or campesterol that is produced or each fine chemicals that is produced thus.

[0378.0.12.12] can separate by this way and be higher than 50% by weight, favourable is higher than 60%, preferably be higher than 70%, especially preferably be higher than 80%, extremely preferably be higher than 90% plant sterol, the particularly Sitosterolum and/or the campesterol that produce in the method.As required, resulting plant sterol, particularly Sitosterolum and/or campesterol can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.12.12] in one embodiment, plant sterol, particularly Sitosterolum and/or campesterol are the mixtures that comprises one or more each fine chemicals.In one embodiment, each fine chemicals meaning here is meant plant sterol, particularly Sitosterolum and/or campesterol.In one embodiment, plant sterol the meaning here is the mixture of each fine chemicals.

The suitable synthetic parent material of the plant sterol that [0380.0.12.12] obtains in the method for the invention, particularly Sitosterolum and/or campesterol as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises the composition that comprises plant sterol that separation produces or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is plant sterol or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine that the inventive method produces.

[0381.0.0.12] and [0382.0.0.12] sees top paragraph [0381.0.0.0] and [0382.0.0.0] for disclosing of [0381.0.0.12] and [0382.0.0.12] these paragraphs

[0383.0.12.12] is in order to prepare the fine chemicals that contains sterol compound, each fine chemicals particularly of the present invention, can use for example acetyl-CoA _ HMGCoA, mevalonic acid, mevalonic acid 5 phosphoric acid, mevalonic acid 5-tetra-sodium, the isopentyl bisphosphate, 5-pyrophosphomevalonic acid, isopentyl tetra-sodium (PIP), dimethyl allene tetra-sodium (DMAPP), PIP+DMAPP, geranylpyrophosphate+IPP, farnesylpyrophosphate, 2 farnesylpyrophosphates, squalene and squalene epoxide, cycloartenol is as the source organic compound, and contain lipid acid and (for example have C in order to prepare the ester that contains plant sterol of the present invention, can to use ₁₀--C ₁₆The lipid acid of carbon atom main chain) oils, fat and/or lipid and/or little organic acid such as acetate, propionic acid or butyric acid are as precursor compound.

Above [0384.0.0.12] sees for disclosing of this paragraph [0384.0.0.0]

The fermented liquid that [0385.0.12.12] obtains with this kind approach, particularly contain fermented liquid with other compound, particularly other sterol or VITAMIN (for example carotenoid such as astaxanthin) or lipid acid blended plant sterol, particularly Sitosterolum and/or campesterol, perhaps contain and comprise and the fermented liquid of the microorganism of other compound such as VITAMIN blended plant sterol, particularly Sitosterolum and/or campesterol or microorganism part (as plastid), dry matter content accounts for 7.5 to 25% of weight usually.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means that the concentration that can utilize sugar in the fermention medium during this period of time remains on or be reduced to 0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

Because plant sterol is usually located at film or plastid, therefore in one embodiment, when by separation method such as centrifugal, filtration, decant, condense/flocculate or the combination of these methods from the fermented liquid kind completely or partially during the separating bio amount, avoiding cell is favourable by filtration.The biomass of doing directly can be added in the animal-feed, as long as the enough height and do not have toxic chemical of the concentration of vitamin-E.

[0386.0.12.12] therefore can also be further purified plant sterol, the particularly Sitosterolum and/or the campesterol that produce according to the present invention.For this reason, the composition that will contain product, the lipid extracts fraction total or part that for example with an organic solvent obtains is as mentioned above carried out saponification (to remove triacylglycerol), distributes (distributing) (from more separating nonpolar epiphase the low phasic property derivative of polar) and separate (by for example open column chromatography, preparative thin-layer chromatography or HPLC) between hexane/methanol, wherein purpose product or impurity are all or part of stays on the chromatography resin (referring to for example Kaluzny etc., JLipid Res 1985; 26:135-140).If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

Paragraph [0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.12] to [0392.0.0.12] saw for disclosing of [0387.0.0.12] to [0392.0.0.12] these paragraphs

[0393.0.12.12] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 is capable, preferred Table I B the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the nucleic acid molecule of 486-491 shown in capable hybridized under lax stringent condition nucleic acid molecule, and randomly separates full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

Paragraph [0394.0.0.0] was to [0399.0.0.0] above [0394.0.0.12] to [0399.0.0.12] saw for disclosing of [0394.0.0.12] to [0399.0.0.12] these paragraphs

Whether [0399.1.12.12] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect and depends on as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or the polypeptide of 486-491 shown in capable or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, 112-117 is capable or 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in the biology phenotype more much at one of activity of proteins.

Paragraph [0400.0.0.0] was to [0416.0.0.0] above [0400.0.0.12] to [0416.0.0.12] saw for disclosing of [0400.0.0.12] to [0416.0.0.12] these paragraphs

[0417.0.12.12] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of plant sterol biosynthetic pathway is had resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block plant sterol in the described biology (particularly each fine chemicals) synthetic inhibitor.

The example of plant sterol synthetic inhibitor or weedicide is for example transferred cyclenes for suppressing the compound of Cytochrome P450 in biological for example microorganism of blocking-up or the plant; Triazole species, for example paclobutrazol or oxole bacterium; Pyridines, for example obtusifoliol; The compound of demethylase inhibitor or inhibition HMG-CoA reductase enzyme is as Mevilonin.

Paragraph [0418.0.0.0] was to [0423.0.0.0] above [0418.0.0.12] to [0423.0.0.12] saw for disclosing of [0418.0.0.12] to [0423.0.0.12] these paragraphs

[0424.0.12.12] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other sterols, plant sterol, carotenoid, VITAMIN or lipid acid.

Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

Paragraph [0425.0.0.0] was to [0430.0.0.0] above [0425.0.0.12] to [0430.0.0.12] saw for disclosing of [0425.0.0.12] to [0430.0.0.12] these paragraphs

[0431.0.12.12] embodiment 1: clone SEQ ID NO:12135 in intestinal bacteria

[0432.0.12.12] uses the method for known good foundation (to consult as Sambrook, J. etc. (1989) " Molecular Cloning:A Laboratory Manual " Cold Spring HarborLaboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Mole-cular Biology ", John Wiley﹠amp; Sons), with SEQ ID NO:12135 be cloned into plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl Acad.Sci.USA, 75:3737-3741); PACYC177 (Change﹠amp; Cohen (1978) J.Bacteriol.134:1141-1156); PBS series plasmid (pBSSK+, pBSSK-and other; Stratagene, LaJolla, USA) or clay such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J.Rosenthal, A., and Waterson, R.H. (1987) Gene 53:283-286) with at expression in escherichia coli.

[0433.0.0.12] and [0434.0.0.12] sees top paragraph [0433.0.0.0] and [0434.0.0.0] for disclosing of [0433.0.0.12] and [0434.0.0.12] these paragraphs

[0435.0.12.12] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.12.12] can be by the plasmid DNA (or other carrier DNAs) that will contain the purpose nucleotide sequence, nucleic acid molecule for example of the present invention or carrier of the present invention or nucleotide sequence change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement green alga (Spongiococcum sp (for example Spongiococcumexentricum) for example, Chlorella certain (Chlorella sp.), haematococcus, Phaeodactylum tricornutum, volvox or Dunaliella salina belong to, cytoalgae PLL 6803, exhibition leaf sword-like leave moss), yeast belong, genus mortierella, the mutagenesis in vivo of Escherichia and above-mentioned other genus is beneficial to produce plant sterol.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

Paragraph [0437.0.5.5] to [0444.0.5.5] and [0441.0.0.0] above [0437.0.5.12] to [0440.0.5.12] and [0441.0.0.12] see for disclosing of [0437.0.5.12] to [0440.0.5.12] and [0441.0.0.12] these paragraphs

[0442.0.5.12], [0443.0.0.12], [0444.0.5.12] and [0445.0.5.12] is for [0442.0.5.12], [0443.0.0.12], top paragraph [0442.0.5.5] is seen in disclosing of [0444.0.5.12] and [0445.0.5.12] these paragraphs, [0443.0.0.0], [0444.0.5.5] and [0445.0.5.5]

Paragraph [0446.0.0.0] to [0450.0.0.0] and [0451.0.5.5] above [0446.0.0.12] to [0450.0.0.12] and [0451.0.5.12] see for disclosing of [0446.0.0.12] to [0450.0.0.12] and [0451.0.5.12] these paragraphs

[0452.0.0.12] is to [0454.0.0.12], [0455.0.5.12] and [0456.0.0.12] for [0452.0.0.12] to [0454.0.0.12], disclosing of [0455.0.5.12] and [0456.0.0.12] these paragraphs seen top [0452.0.0.0] to [0454.0.0.0], [0455.0.5.5] and [0456.0.0.0]

[0457.0.12.12] embodiment 9: the purifying of plant sterol

[0458.0.12.12] analyzes an embodiment of plant sterol: the standard method that is used for these plant sterols is by having flame ionization detector (GC-FID; SAC-5 post, 30m * 0.25mm, 0.25 μ m, not silylated sample) the content of gas Chromatographic Determination plant sterol of the present invention.Another method is to use as the gas chromatography-mass spectrum of above-mentioned post of the same type (GC-MS) and detects.

For by gas chromatographic analysis sterol concentration, can use to be equipped with NB-54 molten silicon capillary column (15m * 0.20mm I.D.; Nordion, Helsinki, Finland) and Hewlett-Packard (HP) 5890 gas-chromatographies of HP 5970A mass detector interface (in electron-bombardment pattern (70eV) operation down).Column oven is set for 10 ℃/minute speed from 230 ℃ to 285 ℃, and sampler and detector are 285 ℃.From sample (200 μ l), extract lipid with chloroform/methanol (2: 1), and carry out transesterify with sodium methylate.(Gylling etc. as described previously, J.Lipid Res 40:593-600,1999), the free sterol that is discharged is a trimethyl silylization, it can use m/z 129 (cholesterol, campesterol and β-Gu Zaichun), m/z 215 (β-sitostanol), m/z 343 (desmosterol), m/z 255 (lathosterol) and m/z 217 (5-α-cholestane by single ion detection technology, internal standard) as selected ion (Vaskonen, Dissertation, Biomedicum Helsinki, on June 19th, 2002) quantitatively.

[0459.0.12.12] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified plant sterol, particularly Sitosterolum and/or campesterol.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

Above [0460.0.0.12] sees for disclosing of this paragraph [0460.0.0.0]

[0461.0.12.12] embodiment 10: clone SEQ ID NO:12135 is used for expressing plant

Above [0462.0.0.12] sees for disclosing of this paragraph [0462.0.0.0]

[0463.0.12.12] passes through pcr amplification SEQ ID NO:12135 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

Paragraph [0464.0.0.0] was to [0466.0.0.0] above [0464.0.0.12] to [0466.0.0.12] saw for disclosing of [0464.0.0.12] to [0466.0.0.12] these paragraphs

[0467.0.12.12] selects following primer sequence for genes of SEQ ID NO:12135:

I) forward primer (SEQ ID NO:12137)

ATGGAACAGAACAGGTTCAAGAAAG

Ii) reverse primer (SEQ ID NO:12138)

TTACAGTTTTTGTTTAGTCGTTTTAAC

Paragraph [0468.0.0.0] was to [0479.0.0.0] above [0468.0.0.12] to [0479.0.0.12] saw for disclosing of [0468.0.0.12] to [0479.0.0.12] these paragraphs

[0480.0.12.12] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:12135

Paragraph [0482.0.0.0] was to [0513.0.0.0] above [0481.0.0.12] to [0513.0.0.12] saw for disclosing of [0481.0.0.12] to [0513.0.0.12] these paragraphs

[0514.0.12.12] selects as another kind of, Heftmann as described, and E. and Hunter, (JChromatogr 1979 for I.R.; 165:283-299) described by HPLC, for example reversed-phase HPLC detects plant sterol.Because the separation principle of HPLC and GC is a complementary, be to prepare the type reversed-phase HPLC earlier so analyze the preferred method of the sterol in the natural product, then resulting sterol fraction is carried out GC-MS and analyze (Bianchini, J.-P. etc.; J Chromatogr 1985; 329:231-246).

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??YER156C	Sitosterolum	??GC	??1.18	??1.39
??YER156C	Campesterol	??GC	??1.17	??1.95
					??YER173W	Campesterol	??GC	??1.23	??1.51
??YKR057W	Sitosterolum	??GC	??1.14	??1.32
					??YKR057W	Campesterol	??GC	??1.20	??1.39
??YOR044W	Campesterol	??GC	??1.28	??1.57
					??YOR084W	Sitosterolum	??GC	??3.59	??3.59
??YPR172W	Campesterol	??GC	??1.17	??1.62
					??b0019	Sitosterolum	??GC	??1.20	??1.47
??b0421	Sitosterolum	??GC	??1.13	??1.52
					??b2699	Sitosterolum	??GC	??1.14	??1.35
??b2699	Campesterol	??GC	??1.17	??1.62
					??b3256	Campesterol	??GC	??1.20	??1.22
??b0050	Sitosterolum	??GC	??1.15	??1.24
					??b0161	Sitosterolum	??GC	??1.12	??1.26

??b0161	Campesterol	??GC	??1.17	??1.42
					??b0464	Campesterol	??GC	??1.16	??1.32
??b1896	Campesterol	??GC	??1.18	??1.59
					??b2341	Campesterol	??GC	??1.18	??1.48
??b2478	Campesterol	??GC	??1.17	??1.37
					??b2822	Campesterol	??GC	??1.21	??1.32
??b4129	Sitosterolum	??GC	??1.21	??1.23

[0515.0.12.12] the 2nd row have shown the plant sterol of being analyzed.The 4th row and the 5th row have shown the ratio of plant sterol between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

Above [0516.0.0.12] sees for disclosing of this paragraph [0516.0.0.0]

[0517.0.12.12] embodiment 14a: the rye grass plant is transformed in the expression of crossing by yeast saccharomyces cerevisiae YOR084W or other Nucleotide of the present invention

Paragraph [0518.0.0.0] was to [0524.0.0.0] above [0518.0.0.12] to [0524.0.0.12] saw for disclosing of [0518.0.0.12] to [0524.0.0.12] these paragraphs

[0525.0.12.12] embodiment 14b: express the soybean transformation plant by crossing of yeast saccharomyces cerevisiae YOR084W or other Nucleotide of the present invention

Paragraph [0526.0.0.0] was to [0529.0.0.0] above [0526.0.0.12] to [0529.0.0.12] saw for disclosing of [0526.0.0.12] to [0529.0.0.12] these paragraphs

[0530.0.12.12] embodiment 14c: maize plant is transformed in the expression of crossing by yeast saccharomyces cerevisiae YOR084W or other Nucleotide of the present invention

Paragraph [0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.12] to [0530.6.0.12] saw for disclosing of [0530.1.0.12] to [0530.6.0.12] these paragraphs

Paragraph [0531.0.0.0] was to [0533.0.0.0] above [0531.0.0.12] to [0533.0.0.12] saw for disclosing of [0531.0.0.12] to [0533.0.0.12] these paragraphs

[0534.0.12.12] embodiment 14d: wheat plant is transformed in the expression of crossing by yeast saccharomyces cerevisiae YOR084W or other Nucleotide of the present invention

Paragraph [0535.0.0.0] was to [0537.0.0.0] above [0535.0.0.12] to [0537.0.0.12] saw for disclosing of [0535.0.0.12] to [0537.0.0.12] these paragraphs

[0538.0.12.12] embodiment 14e: Semen Brassicae campestris/rape plant is transformed in the expression of crossing by yeast saccharomyces cerevisiae YOR084W or other Nucleotide of the present invention

Paragraph [0539.0.0.0] was to [0542.0.0.0] above [0539.0.0.12] to [0542.0.0.12] saw for disclosing of [0539.0.0.12] to [0542.0.0.12] these paragraphs

[0543.0.12.12] embodiment 14f: the alfalfa plant is transformed in the expression of crossing by yeast saccharomyces cerevisiae YOR084W or other Nucleotide of the present invention

Paragraph [0544.0.0.0] was to [0547.0.0.0] above [0544.0.0.12] to [0547.0.0.12] saw for disclosing of [0544.0.0.12] to [0547.0.0.12] these paragraphs

[0548.0.12.12] embodiment 14g: the alfalfa plant is transformed in the expression of crossing by yeast saccharomyces cerevisiae YOR084W or other Nucleotide of the present invention

Paragraph [0549.0.0.0] was to [0552.0.0.0] above [0549.0.0.12] to [0552.0.0.12] saw for disclosing of [0549.0.0.12] to [0552.0.0.12] these paragraphs

Above [0552.2.0.12] sees for disclosing of this paragraph [0552.2.0.0]

[0553.0.12.12]

1. produce the method for plant sterol, particularly Sitosterolum and/or campesterol, it comprises:

(a) raising or generation YER156C, YKR057W, YOR084W, b0019, b0421, b2699, b0050, b0161 and/or b4129 protein and/or YER156C, YER173W, YKR057W, YOR044W, YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or b2822 activity of proteins in non-human being or its one or more parts; With

(b) in allowing described biology, produce under the condition of plant sterol, particularly Sitosterolum and/or campesterol and cultivate this biology.

2. produce the method for plant sterol, particularly Sitosterolum and/or campesterol, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, be respectively 112-117 capable 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide or its fragment, described nucleic acid molecule gives in biology or its part plant sterol, particularly Sitosterolum and/or campesterol quantity increases;

B) contain just like Table I the 5th or 7 row, to be respectively 112-117 capable or 483-485 is capable and/or 118-124 is capable or the nucleic acid molecule of the nucleic acid molecule of 486-491 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of plant sterol, particularly Sitosterolum and/or campesterol quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of plant sterol, particularly Sitosterolum and/or campesterol quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that plant sterol, particularly Sitosterolum in biology or its part and/or campesterol quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, be respectively 112-117 capable 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give in biology or its part plant sterol, particularly Sitosterolum and/or campesterol quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part plant sterol, particularly Sitosterolum and/or campesterol quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively 112-117 capable 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in consensus sequence polypeptide and give biology or its part in plant sterol, particularly Sitosterolum and/or campesterol quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give plant sterol, particularly Sitosterolum and/or the increase of campesterol quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded plant sterol, particularly Sitosterolum and/or campesterol.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded plant sterol, particularly Sitosterolum and/or the campesterol of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, be respectively 112-117 capable 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in polypeptide or its fragment, described nucleic acid molecule gives in biology or its part plant sterol, particularly Sitosterolum and/or campesterol quantity increases;

B) contain just like Table I the 5th or 7 row, to be respectively 112-117 capable or 483-485 is capable and/or 118-124 is capable or the nucleic acid molecule of the nucleic acid molecule of 486-491 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of plant sterol, particularly Sitosterolum and/or campesterol quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of plant sterol, particularly Sitosterolum and/or campesterol quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that plant sterol, particularly Sitosterolum in biology or its part and/or campesterol quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, be respectively 112-117 capable 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give in biology or its part plant sterol, particularly Sitosterolum and/or campesterol quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part plant sterol, particularly Sitosterolum and/or campesterol quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively 112-117 capable 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in consensus sequence polypeptide and give biology or its part in plant sterol, particularly Sitosterolum and/or campesterol quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give plant sterol, particularly Sitosterolum and/or the increase of campesterol quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, be respectively 112-117 capable 483-485 is capable and/or 118-124 is capable or 486-491 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produce or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, sequence shown in the I A.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in plant sterol, particularly Sitosterolum and/or campesterol quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part plant sterol, particularly Sitosterolum and/or campesterol quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps plant sterol, particularly Sitosterolum and/or campesterol level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by plant sterol, the particularly Sitosterolum that will measure and/or campesterol level or expression of polypeptides level and standard plant sterol, the particularly Sitosterolum when described candidate compound or the sample that comprises described multiple compound lack, measured and/or campesterol or expression of polypeptides level; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that plant sterol, particularly Sitosterolum in plant or the microorganism and/or campesterol produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in plant sterol, particularly the polypeptide that increases of Sitosterolum and/or campesterol quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and it is coded and give plant sterol in biology or its part to be provided at the nucleic acid molecule of described read-out system of permission and claim 6, particularly respond compound and described polypeptide bonded detectable signal under the condition of the expression of polypeptides of Sitosterolum and/or campesterol quantity increase; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for the gene product of giving plant sterol in the cell, particularly Sitosterolum and/or campesterol generation raising, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that plant sterol, particularly Sitosterolum and/or campesterol increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce plant sterol, particularly Sitosterolum and/or campesterol;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) plant sterol, particularly Sitosterolum and/or the campesterol level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give plant sterol in the host cell, particularly Sitosterolum and/or the raising of campesterol level after it is expressed with wild-type.

20. identify the method for the gene product of giving plant sterol in the cell, particularly Sitosterolum and/or campesterol generation raising, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that plant sterol, particularly Sitosterolum in biology or its part and/or campesterol quantity or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce plant sterol, particularly Sitosterolum and/or campesterol;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) plant sterol, particularly Sitosterolum and/or the campesterol level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give plant sterol in the host cell, particularly Sitosterolum and/or the raising of campesterol level after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify the nucleic acid molecule of giving plant sterol, particularly Sitosterolum and/or campesterol increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control plant sterol, particularly Sitosterolum and/or campesterol level at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make anti-Sitosterolum and/or the campesterol synthetic weedicide of suppressing of plant.

[0554.0.0.12] summary: see [0554.0.0.0]:

Produce the method for fine chemicals

[0000.0.0.14] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.14] to [0002.0.0.14]: see that [0001.0.0.0] is to [0002.0.0.0]

[0002.1.14.14] L-L-Ala is used for multiple pharmacy and the animal doctor uses.For example, the L-L-Ala with other amino acid be contained in primer solution preparation or parenteral administered formulation as clinical surgery operation before and postoperative food, and as animal feedstuff additive.In addition, L-Ala is used as foodstuff additive because of its sweet taste.

Key component during L-phenylalanine and L-aspartic acid are made as the sweetener aspartame has very important market.Aspartame (Aspartame) (C ₁₄H ₁₈N ₂O ₅), L-aspartyl-L-phenylalanine methyl esters, be the compound of three kinds of compositions, these three kinds of compositions are methyl alcohol, aspartic acid and phenylalanine.The L-aspartic acid is in addition also as seasonings.

Amino acid L-citrulline is the metabolite of ornithine cycle.Other amino acid in this circulation is L-arginine and L-ornithine.The L-citrulline participates in the liver detoxifcation of ammonia, and has shown Ginseng Extract fast.The L-citrulline also has been used for the treatment of ornithine transcarbamylase defective and other urea cycle disorder.In cellular metabolism, L-arginine and L-citrulline can be used as endogenous N source (Ludwig etc., PLANT PHYSIOLOGY, the 101st volume, the 2nd phase, 429-434 page or leaf, 1993).

Glycine is a valuable compounds therefrom, is widely used as foodstuff additive in the processed food and the coarse raw materials in agrochemistry reagent and the medicine.Glycine is the simplest amino acid, and is used for farm crop as the sequestrant of trace nutrient and produces, and nitrogenous fertilizer, at least can on experiment basis.Therefore, glycine is to be used for the representative amino acid that farm crop produce.In fact, whole business-like glycine are produced by building-up process, and for example Strecker is synthetic, the reaction of formaldehyde, ammonium and prussic acid, and the hydrolytic action of resulting amino-nitrile.Glycine is as chelating/complexing agent, plant-growth regulator, the substrate of microbial product, the nitrogenous fertilizer source of positively charged ion nutrient substance.

Serine is the elementary intermediate product in the biosynthesizing of various kinds of cell metabolite, and wherein cell metabolite comprises compound important on the economics for example choline, glycine, halfcystine and tryptophane.In addition, Serine can be used as single carbon donor and passes through and produces 5 from tetrahydrofolate, and the 10-methylene tetrahydrofolate satisfies the 60%-75% of cell to C1 unit aggregate demand.These C1 unit are used for multiple biosynthetic pathway, comprise that methionine(Met), Trophicardyl one phosphoric acid, other purine and some pyrimidines (for example thymidine and hydroxymethylcytosine) are synthetic.

By glycine decarboxylase and the catalytic glycine of serine hydroxymethylase-Serine change, comprise basal metabolism reaction important in the plant by providing a carbon unit to become all biological for many biological respinses.In addition, in plant, this change is the whole portion of photorespiration pathways metabolism, and produces a large amount of photorespiration CO in plastosome ₂(Bauwe etc., Journal ofExperimental Botany, the 54th volume, the 387th phase, 1523-1535 page or leaf, on June 1st, 2003)

The enzymatic change of phenylalanine-tyrosine is known in eukaryotic cell.People's Phenylalanine hydroxylase particular expression in liver so that the L-phenylalanine is converted into L-tyrosine (J.Biol.Chem.269 (12): 9137-46 (1994) such as Wang).The PAH enzymatic defect causes pku, a kind of common inherited disease.

Tyrosine and homoserine and derivative thereof also are used for organic synthesis.For example, tyrosine is chatecolamines or DOPA (dopa), and the precursor of suprarenin, Dopamine HCL and norepinephrine.Multiple beta-amino-γ-ketone acid can prepare from the l-homoserine that commerce can get.

The 5-oxyproline, also called after Pyroglutamic Acid PCA and salt such as PCA sodium salt are used as cosmetic composition, for example hair and skin conditioning agent.A kind of optically active isomer of PCA (L type) is a naturally occurring composition in the mammalian tissues.In addition, the 5-oxyproline is used as template in the homochirality glutamate antagonist is synthetic.

[0003.0.0.14] to [0008.0.0.14]: see that [0003.0.0.0] is to [0008.0.0.0]

[0008.1.14.14] US 5,498,532 disclose the bar shaped bacteria direct fermentation that belongs to Corynebacterium and brevibacterium sp by utilization produces multiple L-amino acid for example L-glutamic acid, glutamine, Methionin, Threonine, Isoleucine, Xie Ansuan, leucine, tryptophane, phenylalanine, tyrosine, Histidine, arginine, ornithine, citrulline and proline(Pro), wherein these bar shaped bacterias innately can not assimilate the lactose of representing carbon source, but can assimilate lactose by recombinant DNA technology.

The another kind of method that produces amino acid such as homoserine is disclosed in US 20010049126, and this method utilization belongs to the bacterium of Escherichia, and this bacterioid contains PTS (phosphotransferase system) gene.Comprise that by auxotroph methane genus bacillus co-production L-glutamic acid and other amino acid Methionin, aspartic acid, L-Ala are described in US 6,110,713.According to US 5,677,156 are taught, and containing the microorganism of aspartase such as brevibacterium flavum AB-41 bacterial strain (FERM BP-1498) and intestinal bacteria ATCC 11303 by adding can produce the L-aspartic acid from toxilic acid or fumaric acid effectively.US 5,354, and 672 disclose by DNA inversion gene integration,temporal being gone into host cell (Bacillus coli cells) and induce aminos secretion, thereby produce the method for tyrosine, methionine(Met) or phenylalanine.It also is known producing citrulline as the product of glutamine metabolism in small intestine or in the arginine biosynthetic pathway, and ornithine transcarbamylase catalysis at this moment produces citrulline from carbamyl phosphate and ornithine.Benninghoff etc. disclose the scavenger cell of handling by interferon-and have produced citrulline and ornithine (InternationalImmunology, the 3rd volume, 413-417 page or leaf, 1991).Disclose the method that produces glycine among the US 20030040085, it comprises: the glycinonitrile aqueous solution is placed at the reaction that is hydrolyzed in the hydrolysising reacting system under the microbial enzyme effect, thus glycinonitrile is converted into glycine, follow the byproduct ammonium.US 20040157290 discloses and has prepared the method that Serine enriches exogenous protein, comprises cultivating the bacterium that comprises cysteine synthase (cysK) gene and the proteinic gene of encoding exogenous.US 20030079255 discloses by importing host microorganism from the gene of the coding phenylalanine deaminase of chromobacterium violaceum (C.violaceum) or rhodotorula glutinis tyrosine and has produced p-Coumaric Acid, and also produces tyrosine and styracin as intermediate product.Produce single cell protein and selected amino acid is known by microbial fermentation, for example US 4,652,527.The seed amino acid with industrial-scale production is a Methionin, sees Tosaka etc., Trends inBiotechnology, 1:70-74 (1983), Tosaka and Takinami, Progress inIndustrial Microbiology, the 24th chapter, 152-172 page or leaf (Aida etc., 1986).Another example is a L-glutamic acid, and its bacterium that has utilized Corynebacterium, brevibacterium sp, Microbacterium (Microbacterium) and Arothrobacter genus is by producing at molasses and the top fermentation of starch hydroxylate.Aspartic acid and L-Ala are produced from fumaric acid and ammonium by the enzymatic mode.The Bacillaceae species have been used for fermenting process to produce amino acid, Tosaka etc.; Tosaka and Takinami, as above name.

[0009.0.14.14] as mentioned above, amino acid is that people and many animals are necessary, for example is used for other application of domestic animal or sanitation and health-care field.The L-aspartic acid is to be difficult to one of amino acid that directly produces by fermentation.Therefore catalysis is at present added ammonia the enzyme of fumaric acid to for commercial applications in producing aspartic acid.As sequestrant, amino acid can improve the biological effectiveness of different metal.Sequestrant can strengthen nutrition usually to be taken in, if but also there is the absorption that can also improve toxic metal in toxic metal.If cation impurity is present in the trace nutrient source (for example cadmium), will make those pollutents be assimilated by plant than the form that is difficult to more utilize is easier to the sequestering action of those metals.

[0010.0.0.14] to [0011.0.0.14]: see that [0010.0.0.0] is to [0011.0.0.0]

[0012.0.14.14] therefore the objective of the invention is to research and develop inexpensive 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine biosynthetic means.Amino acid (depending on organism) is one of restricted composition that the frequency of occurrences is the highest in food or the feed.

[0013.0.0.14] sees [0013.0.0.00]

[0014.0.14.14] therefore, in first embodiment, the present invention relates to produce the method for fine chemicals, fine chemicals is 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine thus.Therefore, in the present invention, term " fine chemicals " such as in the literary composition use relate to " 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine ".In addition, term " fine chemicals " such as in the literary composition use relate to the fine chemicals that comprises 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine.

[0015.0.14.14] in one embodiment, term " fine chemicals " or " each fine chemicals " meaning are meant 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine, the amino acid of the present invention of preferred L configuration, i.e. L-5-oxyproline, L-L-Ala, L-aspartic acid, L-citrulline, L-glycine, L-homoserine, L-phenylalanine, L-Serine and/or L-tyrosine.In whole specification sheets, term " fine chemicals " meaning is meant free form or is bonded to 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or the tyrosine of protein form, the amino acid of the present invention of preferred L configuration, its salt, ester or acid amides.In preferred embodiments, term " fine chemicals " meaning is meant free form or is bonded to L-5-oxyproline, L-L-Ala, L-aspartic acid, L-citrulline, L-glycine, L-homoserine, L-phenylalanine, L-Serine and/or L-tyrosine or its salt of protein form.

[0016.0.14.14] therefore the present invention relates to produce the method for each fine chemicals, comprising:

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YDL127W, b1896, b0161, b0376, b0970, b1343 and/or b3172 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 126-127 and/or 492-496 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. 5-oxyproline or comprise under the condition of fine chemicals of 5-oxyproline and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YIL150C, YFL050C, YER173W, YBL015W, b3008, b2095, b0236, b0486, b1343, bl863, b2489, b2576, b3231 and/or b3767 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 128-133 and/or 497-504 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. L-Ala or comprise under the condition of fine chemicals of L-Ala and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YIL150C, YGR104C, YER173W, b1896, b0730, b0161, b0577, b1343, b2023, b2507, b2576, b2753, b3116, b3169, b3172, b4129 and/or b4346 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 134-138 and/or 505-516 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. aspartic acid or comprise under the condition of fine chemicals of aspartic acid and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YOR245C, YLL013C, b0057, b0462, b1275, b1360, b2239, b2414, b2426, b2489, b3160, b3241, b3926, b4214 and/or b4269 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 139-140 and/or 517-529 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. citrulline or comprise under the condition of fine chemicals of citrulline and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YOL123W, YFL050C, b0057, b0577, b2414, b2489, b2553 and/or b2576 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 141-142 and/or 530-535 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. glycine or comprise under the condition of fine chemicals of glycine and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YEL046C and/or b3767 protein or have the protein of the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, the 143rd and/or 536 row; With

(b) in allowing described biology, produce fine chemicals, i.e. homoserine or comprise under the condition of fine chemicals of homoserine and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YPR138C, YJL072C, YHR130C, YGR101W, YER173W, YAL049C, b3462, b3256, b1886, b1827, b1708, b1697, b0695, b0161, b1228, b2078, b2414, b2576, b2796, b3919, b3938 and/or b3983 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 144-156 and/or 537-545 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. phenylalanine or comprise under the condition of fine chemicals of phenylalanine and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YOR261C, YLR082C, YLL009C, YKR057W, YIL150C, YER152C, YEL045C, b3462, b1886, b1829, b0057, b0486, b2414, b2489, b2664, b3064, b3116, b3160 and/or b3231 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 157-166 and/or 546-554 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. Serine or comprise under the condition of fine chemicals of Serine and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, increase or produce one or more following activity of proteins: YOR350C, YIL150C, YHR130C, YFL050C, b1829, b1827, b0970, b2491 and/or b3983 protein or have protein as the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 167-172 and/or 555-557 are capable; With

(b) in allowing described biology, produce fine chemicals, i.e. tyrosine or comprise under the condition of fine chemicals of tyrosine and make biological growth.

Therefore, the present invention relates to produce the method for fine chemicals, it comprises

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, 126-172 is capable and/or 492-557 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 the row, 126-172 is capable and/or 492-557 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce fine chemicals, particularly make biological growth under the condition of 5-oxyproline and/or L-Ala and/or aspartic acid and/or citrulline and/or glycine and/or homoserine and/or phenylalanine and/or Serine and/or tyrosine.

[0016.1.14.14] therefore, term " fine chemicals " looks like in one embodiment and is meant " 5-oxyproline " or its homologue relevant with Table I-IV 126-127 and/or the capable listed full sequence of 492-496, and the meaning is meant " L-Ala " or its homologue relevant with Table I-IV 128-133 and/or the capable listed full sequence of 497-504 in one embodiment, and the meaning is meant " aspartic acid " or its homologue relevant with Table I-IV 134-138 and/or the capable listed full sequence of 505-516 in one embodiment, and the meaning is meant " citrulline " or its homologue relevant with Table I-IV 139-140 and/or the capable listed full sequence of 517-529 in one embodiment, and the meaning is meant " glycine " or its homologue relevant with Table I-IV 141-142 and/or the capable listed full sequence of 530-535 in one embodiment, and the meaning is meant and Table I-IV the 143rd and/or relevant " homoserine " or its homologue of the 536 listed full sequences of row in one embodiment, and the meaning is meant " phenylalanine " or its homologue relevant with Table I-IV 144-156 and/or the capable listed full sequence of 537-545 in one embodiment, and the meaning is meant " Serine " or its homologue relevant with Table I-IV 157-166 and/or the capable listed full sequence of 546-554 in one embodiment, and the meaning is meant " tyrosine " or its homologue relevant with Table I-IV 167-172 and/or the capable listed full sequence of 555-557 in one embodiment.

Therefore, term " fine chemicals " meaning is meant and Table I-IV the 127th and/or relevant " 5-oxyproline " and " aspartic acid " of the 137 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 128th, 134,161 and/or relevant " L-Ala ", " aspartic acid ", " Serine " and " tyrosine " of the 168 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 129th, 142 and/or relevant " L-Ala ", " glycine " and " tyrosine " of the 170 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 146th and/or relevant " phenylalanine " and " tyrosine " of the 169 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 153rd and/or relevant " phenylalanine " and " tyrosine " of the 172 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 166th and/or relevant " Serine " and " tyrosine " of the 171 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 150th and/or relevant " phenylalanine " and " Serine " of the 164 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 152nd and/or relevant " phenylalanine " and " Serine " of the 165 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 517th, 530 and/or relevant " citrulline ", " glycine " and " Serine " of the 546 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 492nd, 505 and/or relevant " 5-oxyproline ", " aspartic acid " and " phenylalanine " of the 537 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 498th and/or relevant " L-Ala " and " Serine " of the 547 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 531st and/or relevant " glycine " and " aspartic acid " of the 506 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 494th and/or relevant " 5-oxyproline " and " tyrosine " of the 555 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 499th, 495 and/or relevant " L-Ala ", " the 5-oxyproline " and " aspartic acid " of the 507 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 522nd, 532,548 and/or relevant " citrulline ", " glycine ", " Serine " and " phenylalanine " of the 540 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 524th, 501,533 and/or relevant " citrulline ", " L-Ala ", " glycine " and " Serine " of the 549 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 502nd, 535,510 and/or relevant " L-Ala ", " glycine ", " aspartic acid " and " phenylalanine " of the 541 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 552nd and/or relevant " Serine " and " aspartic acid " of the 512 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 525th and/or relevant " citrulline " and " Serine " of the 553 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 496th and/or relevant " 5-oxyproline " and " aspartic acid " of the 514 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 503rd and/or relevant " L-Ala " and " Serine " of the 554 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 504th and/or relevant " L-Ala " and " homoserine " of the 536 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 545th and/or relevant " phenylalanine " and " tyrosine " of the 557 listed full sequences of row in one embodiment;

Term " fine chemicals " meaning is meant and Table I-IV the 130th, 136 and/or relevant " L-Ala ", " aspartic acid " and " phenylalanine " of the 148 listed full sequences of row in one embodiment;

Therefore, term " fine chemicals " can refer to " 5-oxyproline ", " L-Ala ", " aspartic acid ", " citrulline ", " glycine ", " homoserine ", " phenylalanine ", " Serine " and/or " tyrosine " according to environment and context.For the meaning of illustrating term " fine chemicals " is meant " 5-oxyproline ", " L-Ala ", " aspartic acid ", " citrulline ", " glycine ", " homoserine ", " phenylalanine ", " Serine " and/or " tyrosine ", also can use term " each fine chemicals ".

[0017.0.0.14] to [0018.0.0.14]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.14.14] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in the capable and/or 492-557 of 126-172 is capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in the capable and/or 492-557 of 126-172 was capable.

[0020.0.14.14] is surprisingly found out that; At least a such as Table II the 3rd row, for the 126th row of 5-OxoPro and/or for the 128-131 of alanine capable and/or for the 134-136 of aspartic acid capable and/or for the 139-140 of citrulling capable and/or for the 141-142 of glycine capable and/or for the 143rd row of homoserine and/or capable and/or capable and/or capable for the 167-170 of tyrosine for the 157-163 of serine for the 144-149 of phenylalanine shown in saccharomyces cerevisiae protein, and/or at least a such as Table II the 3rd row, for the 127th and/or 492-496 of 5-OxoPro capable and/or for the 132-133 of alanine and/or 497-504 is capable and/or for the 137-138 of aspartic acid and/or 505-516 is capable and/or for the 517-529 of citrulling capable and/or for the 530-535 of glycine capable and/or for the 536th row of homoserine and/or for the 150-156 of phenylalanine and/or 537-545 is capable and/or for the 164-166 of serine and/or 546-554 is capable and/or capable for the 171-172 of tyrosine and/or 555-557 shown in e. coli k12 protein transgene expression in arabidopsis each fine chemicals content of giving institute's conversion of plant increase.

Be surprisingly found out that e. coli k12 protein transgene expression in Arabidopis thaliana is given 5-oxyproline and/or the increase of aspartic acid (or each fine chemicals) content of institute conversion plant shown in Table II the 5th row, the 127th and 137 row.Therefore, in one embodiment, described protein or its homologue are used to produce the 5-oxyproline, and in one embodiment, described protein or its homologue are used to produce aspartic acid; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from 5-oxyproline and/or aspartic acid.

Be surprisingly found out that yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana is given L-Ala and/or aspartic acid and/or Serine and/or the increase of tyrosine (or each fine chemicals) content of institute conversion plant shown in Table II the 5th row, the 128th and 134 and 161 and 168 row.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce aspartic acid, in one embodiment, described protein or its homologue are used to produce Serine, in one embodiment, described protein or its homologue are used to produce tyrosine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or aspartic acid and/or Serine and/or tyrosine.

Be surprisingly found out that the yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 129th and 142 and 170 row is given L-Ala and/or glycine and/or the increase of tyrosine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce glycine, in one embodiment, described protein or its homologue are used to produce tyrosine, in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or glycine and/or tyrosine.

Be surprisingly found out that the yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 146th and 169 row is given phenylalanine and/or the increase of tyrosine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce phenylalanine; In one embodiment, described protein or its homologue are used to produce tyrosine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from phenylalanine and/or tyrosine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 153rd and 172 row is given phenylalanine and/or the increase of tyrosine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce phenylalanine, and in one embodiment, described protein or its homologue are used to produce tyrosine; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from phenylalanine and/or tyrosine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 166th and 171 row is given Serine and/or the increase of tyrosine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce Serine, and in one embodiment, described protein or its homologue are used to produce tyrosine; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from Serine and/or tyrosine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 150th and 164 row is given phenylalanine and/or the increase of Serine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce phenylalanine, and in one embodiment, described protein or its homologue are used to produce Serine; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from phenylalanine and/or Serine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 152nd and 165 row is given phenylalanine and/or the increase of Serine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce phenylalanine, and in one embodiment, described protein or its homologue are used to produce Serine; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from phenylalanine and/or Serine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 517th and 530 and 546 row is given citrulline and/or glycine and/or the increase of Serine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce citrulline; In one embodiment, described protein or its homologue are used to produce glycine, in one embodiment, described protein or its homologue are used to produce Serine, in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from citrulline and/or glycine and/or Serine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 492nd and 505 and 537 row is given 5-oxyproline and/or aspartic acid and/or the increase of phenylalanine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce the 5-oxyproline; In one embodiment, described protein or its homologue are used to produce aspartic acid, in one embodiment, described protein or its homologue are used to produce phenylalanine, in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from 5-oxyproline and/or aspartic acid and/or phenylalanine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 498th and 547 row is given L-Ala and/or the increase of Serine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce Serine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or Serine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 531st and 506 row is given glycine and/or the increase of aspartic acid (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce glycine; In one embodiment, described protein or its homologue are used to produce aspartic acid, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from glycine and/or aspartic acid.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 494th and 555 row is given 5-oxyproline and/or the increase of tyrosine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce the 5-oxyproline; In one embodiment, described protein or its homologue are used to produce tyrosine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from 5-oxyproline and/or tyrosine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 499th and 495 and 507 row is given L-Ala and/or 5-oxyproline and/or the increase of aspartic acid (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce the 5-oxyproline, in one embodiment, described protein or its homologue are used to produce aspartic acid, in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or 5-oxyproline and/or aspartic acid.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 522nd and 532 and 548 and 540 row is given citrulline and/or glycine and/or Serine and/or the increase of phenylalanine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce citrulline; In one embodiment, described protein or its homologue are used to produce glycine, in one embodiment, described protein or its homologue are used to produce Serine, in one embodiment, described protein or its homologue are used to produce phenylalanine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from citrulline and/or glycine and/or Serine and/or phenylalanine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 524th and 501 and 533 and 549 row is given citrulline and/or L-Ala and/or glycine and/or the increase of Serine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce citrulline; In one embodiment, described protein or its homologue are used to produce L-Ala, in one embodiment, described protein or its homologue are used to produce glycine, in one embodiment, described protein or its homologue are used to produce Serine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from citrulline and/or L-Ala and/or glycine and/or Serine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 502nd and 535 and 510 and 541 row is given L-Ala and/or glycine and/or aspartic acid and/or the increase of phenylalanine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce glycine, in one embodiment, described protein or its homologue are used to produce aspartic acid, in one embodiment, described protein or its homologue are used to produce phenylalanine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or glycine and/or aspartic acid and/or phenylalanine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 552nd and 512 row is given Serine and/or the increase of aspartic acid (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce Serine; In one embodiment, described protein or its homologue are used to produce aspartic acid, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from Serine and/or aspartic acid.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 525th and 553 row is given citrulline and/or the increase of Serine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce citrulline; In one embodiment, described protein or its homologue are used to produce Serine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from citrulline and/or Serine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 496th and 514 row is given 5-oxyproline and/or the increase of aspartic acid (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce the 5-oxyproline; In one embodiment, described protein or its homologue are used to produce aspartic acid, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from 5-oxyproline and/or aspartic acid.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 503rd and 554 row is given L-Ala and/or the increase of Serine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce Serine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or Serine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 504th and 536 row is given L-Ala and/or the increase of homoserine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce homoserine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or homoserine.

Be surprisingly found out that the e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 545th and 557 row is given phenylalanine and/or the increase of tyrosine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce phenylalanine; In one embodiment, described protein or its homologue are used to produce tyrosine, and in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from phenylalanine and/or tyrosine.

Be surprisingly found out that the yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 130th and 136 and 148 row is given L-Ala and/or aspartic acid and/or the increase of phenylalanine (or each fine chemicals) content of institute conversion plant.Therefore, in one embodiment, described protein or its homologue are used to produce L-Ala; In one embodiment, described protein or its homologue are used to produce aspartic acid, in one embodiment, described protein or its homologue are used to produce phenylalanine, in one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from L-Ala and/or aspartic acid and/or phenylalanine.

[0021.0.0.14] sees [0021.0.0.0]

The sequence of [0022.0.14.14] yeast saccharomyces cerevisiae YER152C has been published in Dietrich, Nature387 (6632 supplementary issue), and 78-81,1997, and Goffeau, Science 274 (5287), 546-547,1996.As if it have the activity that is similar to tyrosine aminotransferase.Therefore, in one embodiment, the inventive method comprises that as shown here having is similar to the purposes of the active gene product of yeast saccharomyces cerevisiae tyrosine aminotransferase, preferred Xie Ansuan-pyruvic acid transaminase family or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine, particularly increase the quantity of the Serine of free or combining form.In one embodiment, in the methods of the invention, have the active activity of proteins of the tyrosine aminotransferase of being similar to and be enhanced or produce, for example the activity that is similar to the active protein of tyrosine aminotransferase or its homologue from having of yeast saccharomyces cerevisiae is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YAL049C has been published in Bussey etc., Proc.Natl.Acad.Sci.U.S.A.92 (9), and 3809-3813 (1995) and Goffeau, Science 274 (5287), 546-547,1996.Its activity does not characterize yet.Therefore, in one embodiment, the inventive method comprises the gene product with yeast saccharomyces cerevisiae YAL049C protein active as shown here or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase the quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, the YAL049C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YAL049C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b3008 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is PLP dependency cystathionine beta-lyase (beta-Cystathionase) that its activity is defined as, preferred O-succinyl homoserine (sulfydryl)-lyase superfamily.Therefore, in one embodiment, the inventive method comprises that O-succinyl homoserine (sulfydryl) as shown here-lyase superfamily protein (preferably, this protein has PLP dependency cystathionine beta-lyase (beta-Cystathionase) activity) purposes, as shown here its is used for producing each fine chemicals in biological or its part, be L-Ala, preferably free or combining form.In one embodiment, in the methods of the invention, the activity of cystathionine beta-lyase is enhanced or produces, and for example the activity from colibacillary cystathionine beta-lyase or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YOR261C has been published in Dujon etc., Nature 387 (6632 supplementary issue), 98-102 (1997) and Goffeau, Science 274 (5287), 546-547,1996, and its activity to be defined as be that proteasome is regulated the particle subunit, preferred mov-34 protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having mov-34 protein superfamily (preferably, this protein has proteasome and regulates particle subunit activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. Serine particularly increases Serine, the preferred quantity of the Serine of free or combining form.In one embodiment, in the methods of the invention, the activity that proteasome is regulated the particle subunit is enhanced or produces, and for example is enhanced or produces from the proteasome adjusting particle subunit of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YOR245C has been published in Dujon etc.; Nature 387 (6632 supplementary issue); 98-102 (1997) and Goffeau; Science 274 (5287); 546-547; 1996, and its activity to be defined as be acetyl-CoA: Diacrylglycerol acyl transferase, preferred Caenorhabditis elegans (Caenorhabditis elegans) putative protein matter K07B1.4 superfamily.Therefore; in one embodiment; the inventive method comprises as shown here having Caenorhabditis elegans putative protein matter K07B1.4 superfamily (preferably; this protein has acetyl-CoA: the Diacrylglycerol acyl transferase activity) purposes of active gene product or its homologue; as shown here; it is used for producing each fine chemicals in biological or its part, i.e. citrulline particularly increases the quantity of the citrulline of free or combining form.In one embodiment, in the methods of the invention, acetyl-CoA: the activity of Diacrylglycerol acyl transferase is enhanced or produces, and for example from the acetyl-CoA of yeast saccharomyces cerevisiae: the activity of Diacrylglycerol acyl transferase or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YOL123W has been published in Dujon etc., Nature 387 (6632 supplementary issue), 98-102 (1997) and Goffeau, Science 274 (5287), 546-547,1996, and its activity be defined as be participate in premessenger RNA 3 '-shearing and the poly-adenosine factor CF I composition of end processing, preferred heterogeneity ribonucleoprotein HRP1-yeast (yeast saccharomyces cerevisiae) superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having heterogeneity ribonucleoprotein HRP1-yeast (yeast saccharomyces cerevisiae) superfamily (preferably, this protein have participate in premessenger RNA 3 '-shearing of end processing and the activity of poly-adenosine factor CF I composition) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be glycine, particularly increase the quantity of the glycine of free or combining form.In one embodiment, in the methods of the invention, participate in premessenger RNA 3 '-shearing of end processing and the activity of poly-adenosine factor CF I composition be enhanced or produce, for example from the participation premessenger RNA 3 of yeast saccharomyces cerevisiae '-shearing of end processing and the activity of poly-adenosine factor CF I composition or its homologue be enhanced or produce.

The sequence of yeast saccharomyces cerevisiae YLR082C has been published in Johnston, Nature 387 (6632 supplementary issue), 87-90, (1997) and Goffeau, Science 274 (5287), 546-547,1996, and Goffeau, Science 274 (5287), 546-547,1996, and its activity to be defined as be the invalid lethal arrestin of Rad53.Therefore, in one embodiment, the inventive method comprises the purposes with the Rad53 active gene product of invalid lethal arrestin or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine, particularly increase the quantity of the Serine of free or combining form.In one embodiment, in the methods of the invention, the activity of the invalid lethal arrestin of Rad53 is enhanced or produces, and for example is enhanced or produces from the invalid lethal arrestin of Rad53 of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0695 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is sensibility histidine kinase in the two-pack regulator control system that its activity is defined as, preferred sensibility histidine kinase homology superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having sensibility histidine kinase homology superfamily (preferably from colibacillary, this protein has the sensibility histidine kinase activity in the two-pack regulator control system) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. phenylalanine particularly increases the quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, the activity of the sensibility histidine kinase in the two-pack regulator control system is enhanced or produces, and for example the activity from the sensibility histidine kinase in the colibacillary two-pack regulator control system or its homologue is enhanced or produces.

The sequence of e. coli k12 b0730 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is that succinic thiokinase operon transcriptional and/or acyl are replied regulatory gene that its activity is defined as, preferred transcriptional GntR superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having transcriptional GntR superfamily (preferably from colibacillary, this protein has succinic thiokinase operon transcriptional and/or acyl is replied the regulatory gene activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase the quantity of the aspartic acid of free or combining form.In one embodiment, in the methods of the invention, the activity that succinic thiokinase operon transcriptional and/or acyl are replied regulatory gene is enhanced or produces, and for example the activity of replying regulatory gene or its homologue from colibacillary succinic thiokinase operon transcriptional and/or acyl is enhanced or produces.

The sequence of e. coli k12 b1697 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is to have that the ETFP adenine nucleotide-in conjunction with the electron transfer flavoprotein subunit in spline structure territory, preferred electron shifts flavoprotein β chain superfamily that its activity is defined as.Therefore, in one embodiment, the inventive method comprises as shown here having electron transfer flavoprotein β chain superfamily (preferably from colibacillary, this protein has and has the ETFP adenine nucleotide-in conjunction with the activity of the electron transfer flavoprotein subunit in spline structure territory) and the purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase the quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, has ETFP adenine nucleotide-be enhanced or produce, for example from the colibacillary ETFP of having adenine nucleotide-be enhanced or produce in conjunction with the electron transfer flavoprotein subunit in spline structure territory or the activity of its homologue in conjunction with the activity of the electron transfer flavoprotein subunit in spline structure territory.

The sequence of e. coli k12 b1708 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be lipoprotein, preferred conservative putative protein matter HI1314 superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having conservative putative protein matter HI1314 superfamily (preferably from colibacillary, this protein has the lipoprotein activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. phenylalanine particularly increases the quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, the activity of lipoprotein is enhanced or produces, and for example the activity from colibacillary lipoprotein or its homologue is enhanced or produces.

The sequence of e. coli k12 b1827 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is to have the repressor protein (IclR family) of DNA in conjunction with wing spirane structure territory, preferred acetate operon repressor superfamily that its activity is defined as.Therefore, in one embodiment, the inventive method comprises as shown here having acetate operon repressor superfamily (preferably from colibacillary, this protein has and has the activity of DNA in conjunction with the transcription repressor (IclR family) in wing spirane structure territory) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine and/or tyrosine, particularly increase phenylalanine and/or tyrosine, preferred free or the phenylalanine of combining form and/or the quantity of tyrosine.In one embodiment, in the methods of the invention, have DNA and be enhanced or produce, for example be enhanced or produce in conjunction with the transcription repressor protein (IclR family) in wing spirane structure territory or the activity of its homologue from the colibacillary DNA of having in conjunction with the activity of the transcription repressor protein (IclR family) in wing spirane structure territory.

The sequence of e. coli k12 b1829 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the preferred heat shock protein htpX of the heat shock protein superfamily with protease activity.Therefore, in one embodiment, the inventive method comprises as shown here having heat shock protein htpX superfamily (preferably from colibacillary, this protein has the activity of the heat shock protein with protease activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine and/or tyrosine, particularly increase Serine and/or tyrosine, preferably free or the Serine of combining form and/or the quantity of tyrosine.In one embodiment, in the methods of the invention, the activity with heat shock protein of proteolytic enzyme is enhanced or produces, and for example has the heat shock protein of proteolytic enzyme or the activity of its homologue is enhanced or produces from colibacillary.

The sequence of e. coli k12 b1886 has been published in Blattner, Science 277 (5331), 1453-1474,1997, and its activity be defined as be and its activity to be defined as be methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter acceptor, preferable methyl acceptor chemotactic protein matter superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having methyl acceptor chemotactic protein matter superfamily (preferably from colibacillary, this protein has methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter receptor active) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine and/or phenylalanine, particularly increase Serine and/or phenylalanine, preferably free or the Serine of combining form and/or the quantity of phenylalanine.In one embodiment, in the methods of the invention, the activity of methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter acceptor is enhanced or produces, and for example the activity from colibacillary methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter acceptor or its homologue is enhanced or produces.

The sequence of e. coli k12 b1896 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and it is trehalose-6-phosphate synthase that its activity is defined as, preferred schizosaccharomyces pombe α, α-trehalose-6-phosphate synthase (forming UdP) superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having a schizosaccharomyces pombe α from colibacillary, superfamily (preferably for α-trehalose-6-phosphate synthase (forming UdP), this protein has the trehalose-6-phosphate synthase activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be 5-oxyproline and/or aspartic acid, particularly increase 5-oxyproline and/or aspartic acid, preferred free or the 5-oxyproline of combining form and/or the quantity of aspartic acid.In one embodiment, in the methods of the invention, the activity of trehalose-6-phosphate synthase is enhanced or produces, and for example the activity from colibacillary trehalose-6-phosphate synthase or its homologue is enhanced or produces.

The sequence of e. coli k12 b2095 has been published in Blattner, Science 277 (5331), 1453-1474,1997, and it is tagatose-6-phosphokinase that its activity is defined as, the possible tagatose 6-phosphokinase gatZ superfamily of preferred Escherichia.Therefore, in one embodiment, the inventive method comprise as shown here from colibacillary possible tagatose 6-phosphokinase gatZ superfamily with Escherichia (preferably, this protein has tagatose-6-phosphokinase activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. phenylalanine particularly increases L-Ala, the preferred quantity of the L-Ala of free or combining form.In one embodiment, in the methods of the invention, the activity of tagatose-6-phosphokinase is enhanced or produces, and for example the activity from colibacillary tagatose-6-phosphokinase or its homologue is enhanced or produces.

The sequence of e. coli k12 b3256 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be acetyl CoA carboxylase and/or biotin carboxylase enzyme subunit, preferred biotin carboxylase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the biotin carboxylase superfamily (preferably from intestinal bacteria, this protein has acetyl CoA carboxylase and/or biotin carboxylase enzyme subunit activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. phenylalanine particularly increases phenylalanine, the preferred quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, the activity of acetyl CoA carboxylase and/or biotin carboxylase enzyme subunit is enhanced or produces, and for example the activity from colibacillary acetyl CoA carboxylase and/or biotin carboxylase enzyme subunit or its homologue is enhanced or produces.

The sequence of e. coli k12 b3462 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the conformity membrane cell division protein, preferred cell split protein ftsX superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having cell division protein ftsX superfamily (preferably from colibacillary, this protein has conformity membrane cell division protein activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine and/or phenylalanine, particularly increase Serine and/or phenylalanine, preferably free or the Serine of combining form and/or the quantity of phenylalanine.In one embodiment, in the methods of the invention, the activity of conformity membrane cell division protein is enhanced or produces, and for example the activity from colibacillary conformity membrane cell division protein or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YBL015W has been published in Goffeau, Science 274 (5287), 546-547,1996 and Feldmann, EMBO J., 13,5795-5809,1994, and its activity to be defined as be glycoprotein and/or the acetyl-CoA lytic enzyme that contains seminose, preferred acetyl-CoA lytic enzyme superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having acetyl-CoA lytic enzyme superfamily (preferably, this protein has the glycoprotein that contains seminose and/or the activity of acetyl-CoA lytic enzyme) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. L-Ala particularly increases L-Ala, the preferred quantity of the L-Ala of free or combining form.In one embodiment, in the methods of the invention, contain the glycoprotein of seminose and/or the activity of acetyl-CoA lytic enzyme and be enhanced or produce, for example be enhanced or produce from the glycoprotein that contains seminose of yeast saccharomyces cerevisiae and/or the activity of acetyl-CoA lytic enzyme or its homologue.

The sequence of yeast saccharomyces cerevisiae YDL127W has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity to be defined as be G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue).Therefore, in one embodiment, the inventive method comprises the purposes with G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) active gene product or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be the 5-oxyproline, particularly increase 5-oxyproline, the preferred quantity of the 5-oxyproline of free or combining form.In one embodiment, in the methods of the invention, the activity of G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) is enhanced or produces, and for example the G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) or the activity of its homologue from yeast saccharomyces cerevisiae is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YEL045C has been published in Dietrich, Nature 387 (6632 supplementary issue), 78-81,1997.Its activity does not characterize as yet.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product with YEL045C protein active of yeast saccharomyces cerevisiae or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine, particularly increase Serine, the preferred quantity of the Serine of free or combining form.In one embodiment, in the methods of the invention, the YEL045C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YEL045C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YLL009C has been published in Johnston etc., Nature387 (6632 supplementary issue), 87-90,1997 and Goffeau, Science 274 (5287), 546-547,1996, and its activity to be defined as be cytochrome c oxidase copper chaperone.Therefore, in one embodiment, the inventive method comprises the purposes with the active gene product of cytochrome c oxidase copper chaperone or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine, particularly increase Serine, the preferred quantity of the Serine of free or combining form.In one embodiment, in the methods of the invention, the activity of cytochrome c oxidase copper chaperone is enhanced or produces, and for example is enhanced or produces from the cytochrome c oxidase copper chaperone of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YER173W has been published in Dietrich etc., Nature387 (6632 supplementary issue), 78-81,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be to participate in dna damage and reduction division pachytene stage check position activatory check position protein.Therefore, in one embodiment, the inventive method comprises as shown here the have dna damage of participation and the gene product of reduction division pachytene stage check position activatory check position protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala and/or aspartic acid, L-Ala and/or phenylalanine, aspartic acid and/or phenylalanine, L-Ala and/or aspartic acid and/or phenylalanine particularly increase one or both or the whole amino acid be selected from L-Ala and/or aspartic acid and/or phenylalanine, one or both or the whole amino acid whose quantity that are selected from L-Ala and/or aspartic acid and/or phenylalanine of preferred free or combining form.In one embodiment, in the methods of the invention, participate in dna damage and reduction division pachytene stage check position activatory check position activity of proteins and be enhanced or produce, for example be enhanced or produce from the participation dna damage of yeast saccharomyces cerevisiae and the activity of reduction division pachytene stage check position activatory check position protein or its homologue.

The sequence of yeast saccharomyces cerevisiae YFL050C has been published in Murakami etc., Nat.Genet.10 (3), 261-268,1995 and Goffeau etc., Science 274 (5287), 546-547,1996, and it is divalence and/or trivalent inorganic cation transporter, preferably magnesium and cobalt translocator superfamily that its activity is defined as.Therefore, in one embodiment, the inventive method comprises as shown here having magnesium and cobalt translocator superfamily (preferably, this protein has divalence and/or trivalent inorganic cation transporter activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala and/or glycine, L-Ala and/or tyrosine, glycine and/or tyrosine, L-Ala and/or glycine and/or tyrosine particularly increase one or both or the whole amino acid be selected from L-Ala and/or glycine and/or tyrosine, one or both or the whole amino acid whose quantity that are selected from L-Ala and/or glycine and/or tyrosine of preferred free or combining form.In one embodiment, in the methods of the invention, the activity of divalence and/or trivalent inorganic cation transporter is enhanced or produces, and for example is enhanced or produces from the divalence of yeast saccharomyces cerevisiae and/or the activity of trivalent inorganic cation transporter or its homologue.

The sequence of yeast saccharomyces cerevisiae YGR101W has been published in Tettelin etc., Nature387 (6632 supplementary issue), 81-84 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be flat rhombus proteolytic enzyme.Therefore, in one embodiment, the inventive method comprises the gene product with flat rhombus protease activity as shown here or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase phenylalanine, the preferred quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, flat rhombus protease activities is enhanced or produces, and for example is enhanced or produces from the flat rhombus proteolytic enzyme of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YGR104C has been published in Thompson etc., Cell 73:1361-1375 (1993), and its activity to be defined as be yeast rna polymerase II repressor SRB5; And/or RNA polymerase B repressor SRB5, preferred rna plymerase ii repressor SRB5 superfamily.Therefore, in one embodiment, the inventive method comprises the rna plymerase ii repressor SRB5 superfamily that has as shown here, and (preferably, this protein has yeast rna polymerase II repressor SRB5; And/or RNA polymerase B repressor SRB5 activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase aspartic acid, the preferred quantity of the aspartic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of yeast rna polymerase II repressor SRB5 and/or RNA polymerase B repressor SRB5 is enhanced or produces, and for example is enhanced or produces from the yeast rna polymerase II repressor SRB5 of yeast saccharomyces cerevisiae and/or the activity of RNA polymerase B repressor SRB5 or its homologue.

The sequence of sequence YHR130C yeast saccharomyces cerevisiae has been published in Johnston etc., Science265:2077-2082 (1994).Its activity does not characterize as yet.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product with YHR130C protein active of yeast saccharomyces cerevisiae or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine and/or tyrosine, particularly increase phenylalanine and/or tyrosine, preferably free or the phenylalanine of combining form and/or the quantity of tyrosine.In one embodiment, in the methods of the invention, the YHR130C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YHR130C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Churcher etc., Nature387 (6632 supplementary issue), 84-87 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be that the S phase (DNA synthetic) is initial or finish necessary chromobindins.Therefore, in one embodiment, the inventive method comprises that as shown here having the S phase (DNA synthetic) is initial or finishes the purposes of the active gene product of necessary chromobindins or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala and aspartic acid, L-Ala and Serine, L-Ala and tyrosine, aspartic acid and Serine, aspartic acid and tyrosine, Serine and tyrosine, L-Ala and aspartic acid and Serine, L-Ala and aspartic acid and tyrosine, L-Ala and Serine and tyrosine, aspartic acid and Serine and tyrosine and/or L-Ala and aspartic acid and Serine and tyrosine particularly increase L-Ala and aspartic acid, L-Ala and Serine, L-Ala and tyrosine, aspartic acid and Serine, aspartic acid and tyrosine, Serine and tyrosine, L-Ala and aspartic acid and Serine, L-Ala and aspartic acid and tyrosine, L-Ala and Serine and tyrosine, aspartic acid and Serine and tyrosine and/or L-Ala and aspartic acid and Serine and tyrosine, the L-Ala and the aspartic acid of preferred free or combining form, L-Ala and Serine, L-Ala and tyrosine, aspartic acid and Serine, aspartic acid and tyrosine, Serine and tyrosine, L-Ala and aspartic acid and Serine, L-Ala and aspartic acid and tyrosine, L-Ala and Serine and tyrosine, the quantity of aspartic acid and Serine and tyrosine and/or L-Ala and aspartic acid and Serine and tyrosine.In one embodiment, in the methods of the invention, activity initial or that finish necessary chromobindins was enhanced or produced the S phase (DNA synthetic), and activity for example initial from the S phase (DNA is synthetic) of yeast saccharomyces cerevisiae or that finish necessary chromobindins or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YJL072C has been published in Goffeau, Science 274 (5287), 546-547 (1996) and Galibert, EMBO is (9) J.15,2031-2049 (1996), and it is the subunit that chromosomal DNA duplicates required GINS complex body that its activity is defined as, the possible membranin YJL072c superfamily of preferably saccharomyces cerevisiae.Therefore, in one embodiment, the inventive method comprises the possible membranin YJL072c superfamily with yeast saccharomyces cerevisiae as shown here (preferably, this protein has the subunit activity that chromosomal DNA duplicates required GINS complex body) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. phenylalanine particularly increases phenylalanine, the preferred quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, the activity that chromosomal DNA duplicates the subunit of required GINS complex body is enhanced or produces, and for example duplicates the subunit of required GINS complex body from the chromosomal DNA of yeast saccharomyces cerevisiae or the activity of its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YKR057W has been published in Goffeau, Science 274 (5287), 546-547 (1996) and Dujon, Nature 369 (6479), 371-378 (1994), and it is to participate in the biological ribosomal protein that is similar to the S21 ribosomal protein that takes place and translate of rrna, preferred rat ribosomal protein 21 superfamilies that its activity is defined as.Therefore, in one embodiment, the inventive method comprises as shown here having rat ribosomal protein 21 superfamilies (preferably, the ribosomal protein activity that is similar to the S21 ribosomal protein that this protein has the biological generation of participation rrna and translates) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine, particularly increase Serine, the preferred quantity of the Serine of free or combining form.In one embodiment, in the methods of the invention, the activity that participates in the ribosomal protein that is similar to the S21 ribosomal protein of generation of rrna biology and translation is enhanced or produces, and for example is enhanced or produces from the biological ribosomal protein that is similar to the S21 ribosomal protein that takes place and translate of the participation rrna of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YLL013C has been published in Johnston, Nature 387 (6632 supplementary issue), 87-90 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be PUF protein families (it is from the member pumilio that found and Fbf name) member.Therefore, in one embodiment, the inventive method comprises the purposes with PUF protein families (its member pumilio and Fbf name from being found) the active gene product of member or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.In one embodiment, in the methods of the invention, PUF protein families (its member pumilio and Fbf name from being found) member's activity is enhanced or produces, and for example is enhanced or produces from PUF protein families (its member pumilio and Fbf name from the being found) member of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YOR350C has been published in Goffeau, and Science 274 (5287), 546-547 (1996) and Dujon, and Nature 369 (6479), and 371-378 (1994) and its activity do not characterize yet.It is yeast saccharomyces cerevisiae MNE1 protein superfamily member seemingly.Therefore, in one embodiment, the inventive method comprises as shown here having yeast saccharomyces cerevisiae MNE1 protein superfamily (preferably, this protein has the YOR350C activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. tyrosine particularly increases tyrosine, the preferred quantity of the tyrosine of free or combining form.In one embodiment, in the methods of the invention, the YOR350C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YOR350C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YPR138C has been published in Goffeau, Science 274 (5287), 546-547 (1996) and Bussey, Nature 387 (6632 supplementary issue), 103-105 (1997), and it is NH4+ transporter subunit that its activity is defined as, preferred ammonium transporter and/or ammonium transporter nrgA superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having ammonium transporter and/or ammonium transporter nrgA superfamily (preferably, this protein has sNH4+ transporter activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, i.e. phenylalanine particularly increases phenylalanine, the preferred quantity of the phenylalanine of free or combining form.In one embodiment, in the methods of the invention, the activity of NH4+ transporter is enhanced or produces, and for example is enhanced or produces from the NH4+ transporter of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YEL046C has been published in Goffeau, Science 274 (5287), 546-547 (1996) and Dietrich, Nature 387 (6632 supplementary issue), 78-81 (1997), and its activity to be defined as be low specificity L-threonine aldolase.Therefore, in one embodiment, the inventive method comprises the purposes with the active gene product of low specificity L-threonine aldolase or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be homoserine, particularly increase homoserine, the preferred quantity of the homoserine of free or combining form.In one embodiment, in the methods of the invention, the activity of low specificity L-threonine aldolase is enhanced or produces, and for example is enhanced or produces from the low specificity L-threonine aldolase of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0057 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b0057 of having protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, it is citrulline, glycine and/or Serine particularly increase citrulline, particularly increase glycine, particularly increase Serine, particularly increase citrulline and glycine, particularly increase citrulline and Serine, particularly increase glycine and Serine, particularly increase citrulline and glycine and Serine, the citrulline of preferred free or combining form, the quantity of glycine and/or Serine.

The sequence of e. coli k12 b0161 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is pericentral siphon serine protease (heat shock protein) that its activity is defined as, preferred Helicobacterium serine protease superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of pericentral siphon serine protease (heat shock protein) or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, it is the 5-oxyproline, aspartic acid and/or phenylalanine particularly increase the 5-oxyproline, particularly increase aspartic acid, particularly increase phenylalanine, particularly increase 5-oxyproline and aspartic acid, particularly increase 5-oxyproline and phenylalanine, particularly increase aspartic acid and phenylalanine, particularly increase 5-oxyproline and aspartic acid and phenylalanine, the 5-oxyproline of preferred free or combining form, the quantity of aspartic acid and/or phenylalanine.

The sequence of e. coli k12 b0236 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the peptide chain release factor homologue, preferably translate the releasing hormone superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of peptide chain release factor homologue or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala, particularly increase L-Ala, the preferred quantity of the L-Ala of free or combining form.

The sequence of e. coli k12 b0376 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is protein β-Nei Xiananmei/D-alanine carboxypeptidase in conjunction with penicillin that its activity is defined as, preferred intestinal bacteria β-Nei Xiananmei superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the protein β-Nei Xiananmei/active gene product of D-alanine carboxypeptidase in conjunction with penicillin or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be the 5-oxyproline, particularly increase 5-oxyproline, the preferred quantity of the 5-oxyproline of free or combining form.

The sequence of e. coli k12 b0462 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be acridine efflux pump protein, preferred acriflavine resistance protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the gene product of acridine efflux pump protein active or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b0486 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the amino acid/amine transporter (APC family) of supposition, preferred possible membranin ybaT superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having amino acid/amine transporter (APC family) the active gene product of supposition or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala and/or Serine, particularly increase L-Ala, particularly increase Serine, particularly increase L-Ala and Serine, preferably free or the L-Ala of combining form and/or the quantity of Serine.

The sequence of e. coli k12 b0577 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition translocator.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of translocator of supposition or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be glycine and/or aspartic acid, particularly increase glycine, particularly increase aspartic acid, particularly increase glycine and aspartic acid, preferably free or the glycine of combining form and/or the quantity of aspartic acid.

The sequence of e. coli k12 b0970 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be possible glutamate receptor, preferred intestinal bacteria ybhL protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of glutamate receptor of possibility or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be 5-oxyproline and/or tyrosine, particularly increase the 5-oxyproline, particularly increase tyrosine, particularly increase 5-oxyproline and tyrosine, preferably free or the 5-oxyproline of combining form and/or the quantity of tyrosine.

The sequence of e. coli k12 b1228 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b1228 of having protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase phenylalanine, the preferred quantity of the phenylalanine of free or combining form.

The sequence of e. coli k12 b1275 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is the instrumentality (LysR family) of biosynthetic transcriptional of L-halfcystine and sulfur assimilation that its activity is defined as, and preferably regulates protein lysR superfamily.Therefore, in one embodiment, the inventive method comprises (LysR family) the active gene product of the instrumentality with the biosynthetic transcriptional of intestinal bacteria L-halfcystine and sulfur assimilation as shown here or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b1343 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the ATP RNA-dependent helicase that is stimulated by 23S rRNA.Therefore, in one embodiment, the inventive method comprises as shown here having the gene product of the ATP RNA-dependent helicase activity that is stimulated by 23S rRNA or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, it is L-Ala, 5-oxyproline and/or aspartic acid particularly increase L-Ala, particularly increase the 5-oxyproline, particularly increase aspartic acid, particularly increase L-Ala and 5-oxyproline, particularly increase L-Ala and aspartic acid, particularly increase 5-oxyproline and aspartic acid, particularly increase L-Ala and 5-oxyproline and aspartic acid, the L-Ala of preferred free or combining form, the quantity of 5-oxyproline and/or aspartic acid.

The sequence of e. coli k12 b1360 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the dna replication protein matter of supposition, preferred dna replication protein matter dnaC superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of dna replication protein matter of supposition or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b1863 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be that exchange connects restriction endonuclease, preferred dna repair protein matter ruvC superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having a purposes that exchange connects the active gene product of restriction endonuclease or its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala, particularly increase L-Ala, the preferred quantity of the L-Ala of free or combining form.

The sequence of e. coli k12 b2023 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is the glutamine acid amides transferring enzyme subunit of imidzoleglycerol phosphate synthase heterodimer that its activity is defined as, preferred amide transferring enzyme hisH, trpG homology superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of glutamine acid amides transferring enzyme subunit of imidzoleglycerol phosphate synthase heterodimer or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase aspartic acid, the preferred quantity of the aspartic acid of free or combining form.

The sequence of e. coli k12 b2078 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is sensibility histidine kinase in the two-pack regulator control system that its activity is defined as, preferred sensibility histidine kinase homology, envZ protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of sensibility histidine kinase in the two-pack regulator control system or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase phenylalanine, the preferred quantity of the phenylalanine of free or combining form.

The sequence of e. coli k12 b2239 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be Phosphoric acid glycerol esters diester phosphodiesterase.Therefore, in one embodiment, the inventive method comprises as shown here having the gene product of Phosphoric acid glycerol esters diester phosphodiesterase activity or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b2414 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit that its activity is defined as, preferred threonine dehydra(ta)se superfamily.Therefore, in one embodiment, the inventive method comprises the purposes from the colibacillary PLP of having dependent enzyme cysteine synthase A and the active gene product of O-acetylserine sulfhydrylase A subunit or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, it is citrulline, glycine, Serine and/or phenylalanine particularly increase citrulline, particularly increase glycine, particularly increase Serine, particularly increase phenylalanine, particularly increase citrulline and glycine, particularly increase citrulline and Serine, particularly increase citrulline and phenylalanine, particularly increase glycine and Serine, particularly increase glycine and phenylalanine, particularly increase Serine and phenylalanine, particularly increase citrulline and glycine and Serine, particularly increase citrulline and glycine and phenylalanine, particularly increase citrulline and Serine and phenylalanine, particularly increase citrulline and glycine and phenylalanine, particularly increase citrulline and glycine and Serine and phenylalanine, the citrulline of preferred free or combining form, glycine, the quantity of Serine and/or phenylalanine.

The sequence of e. coli k12 b2426 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is the oxydo-reductase with NAD (P) binding domains of supposition that its activity is defined as, preferred ribitol dehydrogenase, short-chain alcohol dehydrogenase homology superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary oxidoreductase activity with NAD (P) binding domains with supposition or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b2489 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be hydrogenase Fe-S subunit, preferred psbG protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of hydrogenase Fe-S subunit or its homologue, as shown here, it is used for producing each fine chemicals, i.e. citrulline in biological or its part, L-Ala, glycine and/or Serine, particularly increase citrulline, particularly increase L-Ala, particularly increase glycine, particularly increase Serine, particularly increase citrulline and L-Ala, particularly increase citrulline and glycine, particularly increase citrulline and Serine, particularly increase L-Ala and glycine, particularly increase L-Ala and Serine, particularly increase glycine and Serine, particularly increase citrulline and L-Ala and glycine, particularly increase citrulline and L-Ala and Serine, particularly increase citrulline and glycine and Serine, particularly increase L-Ala and glycine and Serine, particularly increase citrulline and L-Ala and glycine and Serine, the citrulline of preferred free or combining form, L-Ala, the quantity of glycine and/or Serine.

The sequence of e. coli k12 b2491 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be transcriptional activation agent (EBP family) with σ 54 interactional hydrogenase 4 genetic expressions.Therefore, in one embodiment, the inventive method comprises as shown here having and transcriptional activation agent (EBP family) the active gene product of σ 54 interactional hydrogenase 4 genetic expressions or the purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be tyrosine, particularly increase tyrosine, the preferred quantity of the tyrosine of free or combining form.

The sequence of e. coli k12 b2507 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is GMP synthetic enzyme (glutamine transaminase) that its activity is defined as, preferred GMP synthase (hydrolysis glutamine), trpG homology superfamily.Therefore, in one embodiment, the inventive method comprises the purposes from the colibacillary GMP of having synthetic enzyme (glutamine transaminase) active gene product or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase aspartic acid, the preferred quantity of the aspartic acid of free or combining form.

The sequence of e. coli k12 b2553 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the adjusting albumen P-II of glutamine synthetase, preferably regulate albumen P-II superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of adjusting albumen P-II of glutamine synthetase or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be glycine, particularly increase glycine, the preferred quantity of the glycine of free or combining form.

The sequence of e. coli k12 b2576 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be ATP RNA-dependent helicase.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary ATP of having RNA-dependent helicase activity or the purposes of its homologue, as shown here, it is used for producing each fine chemicals, i.e. L-Ala in biological or its part, glycine, aspartic acid and/or phenylalanine, particularly increase L-Ala, particularly increase glycine, particularly increase aspartic acid, particularly increase phenylalanine, particularly increase L-Ala and glycine, particularly increase L-Ala and aspartic acid, particularly increase L-Ala and phenylalanine, particularly increase glycine and aspartic acid, particularly increase glycine and phenylalanine, particularly increase aspartic acid and phenylalanine, particularly increase L-Ala and glycine and aspartic acid, particularly increase L-Ala and glycine and phenylalanine, particularly increase L-Ala and aspartic acid and phenylalanine, particularly increase glycine and aspartic acid and phenylalanine, particularly increase L-Ala and glycine and aspartic acid and phenylalanine, preferred L-Ala, glycine, free or the combining form of aspartic acid and/or phenylalanine quantity.

The sequence of e. coli k12 b2664 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be supposition have the transcription repressor (GntR family) of DNA, preferred possible transcriptional gabP superfamily in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprise as shown here from colibacillary have a supposition have DNA in conjunction with transcription repressor (GntR family) the active gene product in wing spirane structure territory or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine, particularly increase Serine, the preferred quantity of the Serine of free or combining form.

The sequence of e. coli k12 b2753 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is aminopeptidase during alkaline phosphatase isoenzyme changes that its activity is defined as, and preferred alkaline phosphatase isoenzyme changes the protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the gene product of the amino-peptidase activity of alkaline phosphatase isoenzyme in changing or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase aspartic acid, the preferred quantity of the aspartic acid of free or combining form.

The sequence of e. coli k12 b2796 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is the Serine translocator (HAAAP family) of supposition that its activity is defined as, preferred Threonine-Serine permease superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having Serine translocator (HAAAP family) the active gene product of supposition or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase phenylalanine, the preferred quantity of the phenylalanine of free or combining form.

The sequence of e. coli k12 b3064 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is the sialoglycoprotein endopeptidase with Actin muscle sample ATP enzymatic structure territory of supposition that its activity is defined as, preferred sialoglycoprotein endopeptidase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary sialoglycoprotein endopeptidase activity with Actin muscle sample ATP enzymatic structure territory with supposition or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine, particularly increase Serine, the preferred quantity of the Serine of free or combining form.

The sequence of e. coli k12 b3116 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is the L-Threonine/L-Serine permease (HAAAP family) of anaerobic induction that its activity is defined as, preferred Threonine-Serine permease superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having L-Threonine/L-Serine permease (HAAAP family) the active gene product of anaerobic induction or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be Serine and/or aspartic acid, particularly increase Serine, particularly increase aspartic acid, particularly increase Serine and aspartic acid, preferably free or the Serine of combining form and/or the quantity of aspartic acid.

The sequence of e. coli k12 b3160 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the monooxygenase with luciferase sample atpase activity of supposition, preferred ynbW protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary monooxygenase activity with luciferase sample atpase activity with supposition or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline and/or Serine, particularly increase citrulline, particularly increase Serine, particularly increase citrulline and Serine, preferably free or the citrulline of combining form and/or the quantity of Serine.

The sequence of e. coli k12 b3169 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be Transcription Termination-antitermination factor, preferred intestinal bacteria transcription factor nusA superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of Transcription Termination-antitermination factor or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase aspartic acid, the preferred quantity of the aspartic acid of free or combining form.

The sequence of e. coli k12 b3172 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be arginyl succsinic acid synthetic enzyme, preferred arginyl succsinic acid synthase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the gene product of arginyl succsinic acid synthase activity or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be 5-oxyproline and/or aspartic acid, particularly increase the 5-oxyproline, particularly increase aspartic acid, particularly increase 5-oxyproline and aspartic acid, preferably free or the 5-oxyproline of combining form and/or the quantity of aspartic acid.

The sequence of e. coli k12 b3231 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L13, preferred intestinal bacteria ribosomal protein L13 superfamily.Therefore, in one embodiment, the inventive method comprises the purposes from the colibacillary active gene product of the 50S of having ribosomal subunit protein matter L13 or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala and/or Serine, particularly increase L-Ala, particularly increase Serine, particularly increase L-Ala and Serine, preferably free or the L-Ala of combining form and/or the quantity of Serine.

The sequence of e. coli k12 b3241 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the localized multi-medicine resistance protein of film.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary localized multi-medicine resistance protein active of film with supposition or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b3767 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the big subunit of acetolactate synthase isozyme II.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of the big subunit of acetolactate synthase isozyme II or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be L-Ala and/or homoserine, particularly increase L-Ala, particularly increase homoserine, particularly increase L-Ala and homoserine, preferably free or the L-Ala of combining form and/or the quantity of homoserine.

The sequence of e. coli k12 b3919 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be triosephosphate isomerase, preferably phosphoric acid triose isomerase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of triosephosphate isomerase or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase phenylalanine, the preferred quantity of the phenylalanine of free or combining form.

The sequence of e. coli k12 b3926 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be glycerol kinase, preferred xylulokinase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of glycerol kinase or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b3938 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the biosynthetic transcription repressor of methionine(Met), preferred metJ protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of the biosynthetic transcription repressor of methionine(Met) or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine, particularly increase phenylalanine, the preferred quantity of the phenylalanine of free or combining form.

The sequence of e. coli k12 b3983 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L12, preferred intestinal bacteria ribosomal protein L11 superfamily.Therefore, in one embodiment, the inventive method comprises the purposes from the colibacillary active gene product of the 50S of having ribosomal subunit protein matter L12 or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be phenylalanine and/or tyrosine, particularly increase phenylalanine, particularly increase tyrosine, particularly increase phenylalanine and tyrosine, preferably free or the phenylalanine of combining form and/or the quantity of tyrosine.

The sequence of e. coli k12 b4129 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be derivable Methionin tRNA synthetic enzyme, preferred Lysine-tRNA ligase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the gene product of derivable Methionin tRNA synthase activity or a purposes of its homologue from colibacillary, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase aspartic acid, the preferred quantity of the aspartic acid of free or combining form.

The sequence of e. coli k12 b4214 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be ammonium movement system structural protein, preferred Aquifex aeolicus cysQ protein superfamily.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the active gene product of ammonium movement system structural protein or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b4269 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be supposition have NAD (P) binding domains and a GroES structural domain alcoholdehydrogenase.Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary purposes with NAD (P) binding domains and the active gene product of GroES structural domain alcoholdehydrogenase or its homologue with supposition, as shown here, it is used for producing each fine chemicals in biological or its part, be citrulline, particularly increase citrulline, the preferred quantity of the citrulline of free or combining form.

The sequence of e. coli k12 b4346 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and it is 5-methylcytosine specificity restriction enzyme McrBC composition that its activity is defined as, preferred 5-methylcytosine specificity restriction enzyme B ultrasonic family.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary purposes with the 5-methylcytosine specificity restriction enzyme active gene product of McrBC composition or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be aspartic acid, particularly increase aspartic acid, the preferred quantity of the aspartic acid of free or combining form.

The homologous compound (=homologue) of [0023.0.14.14] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.

In one embodiment, shown in Table II the 3rd row, the 126th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred 5-hydroxyproline content increases.In one embodiment, homologue is the homologue that has sequence shown in Table I or II the 7th row, the 126th row respectively.In one embodiment, shown in Table II the 3rd row, the 126th row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 126th row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 126th row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 126th row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 126th row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 126th row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 126th row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively shown in the 128th, 129,130 or 131 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred L-Ala content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row respectively, is respectively sequence shown in the 128th, 129,130 or 131 row.In one embodiment, Table II the 3rd row, be respectively shown in the 128th, 129,130 or 131 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 128th, 129,130 or 131 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 128th, 129,130 or 131 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 128th, 129,130 or 131 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 128th, 129,130 or 131 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 128th, 129,130 or 131 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 128th, 129,130 or 131 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively shown in the 134th, 135 or 136 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred aspartate content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row respectively, is respectively sequence shown in the 134th, 135 or 136 row.In one embodiment, Table II the 3rd row, be respectively shown in the 134th, 135 or 136 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 134th, 135 or 136 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 134th, 135 or 136 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 134th, 135 or 136 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 134th, 135 or 136 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 134th, 135 or 136 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 134th, 135 or 136 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively shown in the 139th or 140 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred citrulline content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row respectively, is respectively sequence shown in the 139th or 140 row.In one embodiment, Table II the 3rd row, be respectively shown in the 139th or 140 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 139th or 140 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 139th or 140 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 139th or 140 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 139th or 140 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 139th or 140 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 139th or 140 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively shown in the 141st or 142 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred glycine content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row respectively, is respectively sequence shown in the 141st or 142 row.In one embodiment, Table II the 3rd row, be respectively shown in the 141st or 142 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 141st or 142 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 141st or 142 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 141st or 142 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 141st or 142 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 141st or 142 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 141st or 142 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II the 3rd row, the 143rd row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred homoserine content increases.In one embodiment, homologue is the homologue that has sequence shown in Table I or II the 7th row, the 143rd row respectively.In one embodiment, shown in Table II the 3rd row, the 143rd row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 143rd row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 143rd row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 143rd row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 143rd row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 143rd row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 143rd row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively shown in the 144th, 145,146,147,148 or 149 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred phenylalanine content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row respectively, is respectively sequence shown in the 144th, 145,146,147,148 or 149 row.In one embodiment, Table II the 3rd row, be respectively shown in the 144th, 145,146,147,148 or 149 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 144th, 145,146,147,148 or 149 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 144th, 145,146,147,148 or 149 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 144th, 145,146,147,148 or 149 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 144th, 145,146,147,148 or 149 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 144th, 145,146,147,148 or 149 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 144th, 145,146,147,148 or 149 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively shown in the 157th, 158,159,160,161,162 or 163 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred serine content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row respectively, is respectively sequence shown in the 157th, 158,159,160,161,162 or 163 row.In one embodiment, Table II the 3rd row, be respectively shown in the 157th, 158,159,160,161,162 or 163 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 157th, 158,159,160,161,162 or 163 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 157th, 158,159,160,161,162 or 163 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 157th, 158,159,160,161,162 or 163 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 157th, 158,159,160,161,162 or 163 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 157th, 158,159,160,161,162 or 163 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 157th, 158,159,160,161,162 or 163 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively shown in the 167th, 168,169 or 170 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred tyrosine content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row respectively, is respectively sequence shown in the 167th, 168,169 or 170 row.In one embodiment, Table II the 3rd row, be respectively shown in the 167th, 168,169 or 170 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 167th, 168,169 or 170 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 167th, 168,169 or 170 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 167th, 168,169 or 170 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 167th, 168,169 or 170 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 167th, 168,169 or 170 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 167th, 168,169 or 170 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, be respectively the 127th and/or 492-496 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred 5-hydroxyproline content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively the 127th and/or 492-496 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively the 127th and/or 492-496 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively the 127th and/or 492-496 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively the 127th and/or 492-496 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively the 127th and/or 492-496 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively the 127th and/or 492-496 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively the 127th and/or 492-496 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively 132-133 and/or 497-504 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred L-Ala content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 132-133 and/or 497-504 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively 132-133 and/or 497-504 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively 132-133 and/or 497-504 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively 132-133 and/or 497-504 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively 132-133 and/or 497-504 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively 132-133 and/or 497-504 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively 132-133 and/or 497-504 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively 137-138 and/or 505-516 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred aspartate content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 137-138 and/or 505-516 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively 137-138 and/or 505-516 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively 137-138 and/or 505-516 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively 137-138 and/or 505-516 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively 137-138 and/or 505-516 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively 137-138 and/or 505-516 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively 137-138 and/or 505-516 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively 517-529 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred citrulline content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 517-529 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively 517-529 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively 517-529 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively 517-529 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively 517-529 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively 517-529 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively 517-529 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively 530-535 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred glycine content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 530-535 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively 530-535 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively 530-535 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively 530-535 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively 530-535 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively 530-535 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively 530-535 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, shown in Table II the 3rd row, the 536th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred homoserine content increase.In one embodiment, homologue is the homologue that has Table I or II the 7th row, is respectively sequence shown in the 536th row.In one embodiment, shown in Table II the 3rd row, the 536th row in the polypeptide homologue of any one from bacterium.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 536th row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 536th row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 536th row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 536th row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 536th row is to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively 150-156 and/or 537-545 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred phenylalanine content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 150-156 and/or 537-545 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively 150-156 and/or 537-545 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively 150-156 and/or 537-545 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively 150-156 and/or 537-545 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively 150-156 and/or 537-545 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively 150-156 and/or 537-545 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively 150-156 and/or 537-545 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively 164-166 and/or 546-554 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred serine content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 164-166 and/or 546-554 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively 164-166 and/or 546-554 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively 164-166 and/or 546-554 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively 164-166 and/or 546-554 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively 164-166 and/or 546-554 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively 164-166 and/or 546-554 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively 164-166 and/or 546-554 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively 171-172 and/or 555-557 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred tyrosine content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively 171-172 and/or 555-557 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively 171-172 and/or 555-557 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II the 3rd row, be respectively 171-172 and/or 555-557 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively 171-172 and/or 555-557 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively 171-172 and/or 555-557 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively 171-172 and/or 555-557 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively 171-172 and/or 555-557 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

[0023.1.14.14] as Table II the 3rd row, the homologue that is respectively the polypeptide of the capable and/or 492-557 of 126-172 shown in capable can be by Table I the 7th row, be respectively the capable and/or 492-557 of 126-172 capable shown in nucleic acid molecule encoding polypeptide or can be table lI the 7th row, be respectively the capable and/or 492-557 of 126-172 capable shown in polypeptide.As Table II the 3rd row, the homologue that is respectively the polypeptide of the capable and/or 492-557 of 126-172 shown in capable can be by Table I the 7th row, be respectively the capable and/or 492-557 of 126-172 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, be respectively the capable and/or 492-557 of 126-172 capable shown in polypeptide.

Table II the 3rd row, be respectively 126-127 and/or 492-496 capable shown in the homologue of polypeptide can be have increase the 5-hydroxyproline content and/or quantity is active, by Table I the 7th row, be respectively 126-127 and/or 492-496 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 126-127 and/or 492-496 capable shown in polypeptide.

Table II the 3rd row, be respectively 128-133 and/or 497-504 capable shown in the homologue of polypeptide can be have increase L-Ala content and/or quantity is active, by Table I the 7th row, be respectively 128-133 and/or 497-504 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 128-133 and/or 497-504 capable shown in polypeptide.

Table II the 3rd row, be respectively 134-138 and/or 505-516 capable shown in the homologue of polypeptide can be have increase aspartate content and/or quantity is active, by Table I the 7th row, be respectively 134-138 and/or 505-516 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 134-138 and/or 505-516 capable shown in polypeptide.

Table II the 3rd row, be respectively 139-140 and/or 517-529 capable shown in the homologue of polypeptide can be have increase citrulline content and/or quantity is active, by Table I the 7th row, be respectively 139-140 and/or 517-529 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 139-140 and/or 517-529 capable shown in polypeptide.

Table II the 3rd row, be respectively 141-142 and/or 530-535 capable shown in the homologue of polypeptide can be have increase glycine content and/or quantity is active, by Table I the 7th row, be respectively 141-142 and/or 530-535 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 141-142 and/or 530-535 capable shown in polypeptide.

Table II the 3rd row, the homologue that is respectively polypeptide shown in the 143rd and/or 536 row can be have increase homoserine content and/or quantity is active, by Table I the 7th row, the polypeptide that is respectively nucleic acid molecule encoding shown in the 143rd and/or 536 row or Table II the 7th row, be respectively polypeptide shown in the 143rd and/or 536 row.

Table II the 3rd row, be respectively 144-156 and/or 537-545 capable shown in the homologue of polypeptide can be have increase phenylalanine content and/or quantity is active, by Table I the 7th row, be respectively 144-156 and/or 537-545 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 144-156 and/or 537-545 capable shown in polypeptide.

Table II the 3rd row, be respectively 157-166 and/or 546-554 capable shown in the homologue of polypeptide can be have increase serine content and/or quantity is active, by Table I the 7th row, be respectively 157-166 and/or 546-554 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 157-166 and/or 546-554 capable shown in polypeptide.

Table II the 3rd row, be respectively 167-172 and/or 555-557 capable shown in the homologue of polypeptide can be have increase tyrosine content and/or quantity is active, by Table I the 7th row, be respectively 167-172 and/or 555-557 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, be respectively 167-172 and/or 555-557 capable shown in polypeptide.

[0024.0.0.14] sees [0024.0.0.0]

[0025.0.14.14] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause biology or its part, amino acid levels increases in the preferred described biomass cells, 5-oxyproline particularly, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine level increase, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example Table II the 3rd row, for the 126-127 of 5-oxyproline and/or 492-496 is capable and/or for the 128-133 of L-Ala and/or 497-504 is capable and/or for the 134-138 of aspartic acid and/or 505-516 is capable and/or for the 139-140 of citrulline and/or 517-529 is capable and/or for the 141-142 of glycine and/or 530-535 is capable and/or for the 143rd and/or 536 row of homoserine and/or for the 144-156 of phenylalanine and/or 537-545 is capable and/or for the 157-166 of Serine and/or 546-554 is capable and/or for the 167-172 of tyrosine and/or 555-557 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in proteinic above-mentioned extra activity.

In whole specification sheets, if this kind protein or polypeptide still have the Table II of being respectively the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in any one proteinic biology or the enzyme activity, if promptly with Table II the 3rd row, be respectively the 126th and/or 128-131 and/or 134-136 is capable and/or 139-140 is capable and/or 141-142 is capable and/or the 143rd row and/or 144-149 is capable and/or 157-163 is capable and/or 167-170 capable shown in any one yeast saccharomyces cerevisiae protein compare and/or with Table II the 3rd row, be respectively the 127th and/or 492-496 is capable and/or 132-133 and/or 497-504 is capable and/or 137-138 and/or 505-516 is capable and/or 517-529 is capable and/or 530-535 is capable and/or the 536th row and/or 150-156 and/or 537-545 is capable and/or 164-166 and/or 546-554 is capable and/or 171-172 and/or 555-557 capable shown in any one e. coli k12 protein compare, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.14] to [0033.0.0.14]: see that [0025.1.0.0] is to [0033.0.0.0]

[0034.0.14.14] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, it has Table II the 3rd row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein, or by Table I the 5th row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the protein of nucleic acid molecule encoding, or its homologue is (as Table I the 7th row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in homologue) active protein expression level or active aspect difference, and they are different and therefore show the fine chemicals quantity that increase aspect biological chemistry or genetics reason.

[0035.0.0.14] to [0044.0.0.14]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.14.14] in one embodiment, at yeast saccharomyces cerevisiae protein YAL049C or its homologue, for example under the activity situation about being enhanced of YAL049C protein (shown in Table I the 5th or 7 row, the 149th row), give each fine chemicals, preferred phenylalanine be increased in 30% and 74% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBL015W or its homologue, the glycoprotein and/or the acetyl-CoA lytic enzyme that for example contain seminose, under the situation that the activity of preferred acetyl-CoA lytic enzyme superfamily (shown in Table I the 5th or 7 row, the 131st row) is enhanced, give each fine chemicals, preferred L-Ala be increased in 29% and 204% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDL127W or its homologue, for example under the activity situation about being enhanced of G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) (shown in Table I the 5th or 7 row, the 126th row), give each fine chemicals, preferred 5-oxyproline be increased in 40% and 127% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YEL045C or its homologue, for example under the activity situation about being enhanced of YEL045C protein (shown in Table I the 5th or 7 row, the 163rd row), give each fine chemicals, preferred Serine be increased in 23% and 80% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YEL046C or its homologue, under the situation that the activity of for example low specificity L-threonine aldolase (shown in Table I the 5th or 7 row, the 143rd row) is enhanced, give each fine chemicals, preferred homoserine be increased in 44% and 177% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER152C or its homologue, for example be similar under the activity situation about being enhanced of tyrosine aminotransferase (shown in Table I the 5th or 7 row, the 162nd row), give each fine chemicals, preferred Serine be increased in 23% and 75% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 130th row), give each fine chemicals, preferred L-Ala be increased in 21% and 132% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 136th row), give each fine chemicals, preferred aspartic acid be increased in 64% and 93% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 130th and/or 136 row), give each fine chemicals, preferred L-Ala and aspartic acid be increased in 21% and 132%, preferred 64% and 132% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 148th row), give each fine chemicals, preferred phenylalanine be increased in 44% and 99% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 130th and/or 148 row), give each fine chemicals, preferred L-Ala and phenylalanine be increased in 21% and 132%, preferred 44% and 132% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 136th and/or 148 row), give each fine chemicals, preferred aspartic acid and phenylalanine be increased in 44% and 99%, preferred 64% and 99% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 130th and/or 136 and/or 148 row), give each fine chemicals, preferred L-Ala and aspartic acid and phenylalanine be increased in 21% and 132%, preferred 44% and 132%, more preferably 64% and 132% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, under the activity situation about being enhanced of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 129th row), give each fine chemicals, preferred L-Ala be increased in 19% and 104% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, under the activity situation about being enhanced of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 142nd row), give each fine chemicals, preferred glycine be increased in 36% and 74% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, under the activity situation about being enhanced of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 129th and/or 142 row), give each fine chemicals, preferred L-Ala and glycine be increased in 19% and 104%, preferred 36% and 104% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, under the activity situation about being enhanced of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 170th row), give each fine chemicals, preferred tyrosine be increased in 42% and 56% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, under the activity situation about being enhanced of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 129th and/or 170 row), give each fine chemicals, preferred L-Ala and tyrosine be increased in 19% and 104%, preferred 42% and 104% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, under the activity situation about being enhanced of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 142nd and/or 170 row), give each fine chemicals, preferred glycine and tyrosine be increased in 36% and 74%, preferred 42% and 74% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, under the activity situation about being enhanced of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 129th and/or 142 and/or 170 row), give each fine chemicals, preferred L-Ala and glycine and tyrosine be increased in 19% and 104%, preferred 36% and 104%, more preferably 42% and 104% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YGR101W or its homologue, under the activity situation about being enhanced of for example flat rhombus proteolytic enzyme (shown in Table I the 5th or 7 row, the 147th row), give each fine chemicals, preferred phenylalanine be increased in 40% and 92% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YGR104C or its homologue, under the activity situation about being enhanced of for example flat rhombus proteolytic enzyme (shown in Table I the 5th or 7 row, the 135th row), give each fine chemicals, preferred aspartic acid be increased in 108% and 126% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YHR130C or its homologue, for example under the activity situation about being enhanced of YHR130C protein (shown in Table I the 5th or 7 row, the 146th row), give each fine chemicals, preferred phenylalanine be increased in 77% and 77%, preferred 77% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YHR130C or its homologue, for example under the activity situation about being enhanced of YHR130C protein (shown in Table I the 5th or 7 row, the 169th row), give each fine chemicals, preferred tyrosine be increased in 51% and 126% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YHR130C or its homologue, for example under the activity situation about being enhanced of YHR130C protein (shown in Table I the 5th or 7 row, the 146th and/or 169 row), give each fine chemicals, preferred phenylalanine and tyrosine be increased in 51% and 126%, preferred 77% and 126%, more preferably 126% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th row) is enhanced, give each fine chemicals, preferred L-Ala be increased in 47% and 328% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 134th row) is enhanced, give each fine chemicals, preferred aspartic acid be increased in 308% and 308%, preferred 308% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 134 row) is enhanced, give each fine chemicals, preferred L-Ala and aspartic acid be increased in 47% and 328%, preferred 308% and 328% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 161st row) is enhanced, give each fine chemicals, preferred Serine be increased in 30% and 322% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 161 row) is enhanced, give each fine chemicals, preferred L-Ala and Serine be increased in 30% and 328%, preferred 47% and 328% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 134th and/or 161 row) is enhanced, give each fine chemicals, preferred aspartic acid and Serine be increased in 30% and 322%, preferred 308% and 328% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 134 and/or 161 row) is enhanced, give each fine chemicals, preferred L-Ala and aspartic acid and Serine be increased in 30% and 328%, preferred 47% and 328%, more preferably 308% and 328% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 168th row) is enhanced, give each fine chemicals, preferred tyrosine be increased in 698% and 698%, preferred 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 168 row) is enhanced, give each fine chemicals, preferred L-Ala and tyrosine be increased in 47% and 698%, preferred 698% and 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 134th and/or 168 row) is enhanced, give each fine chemicals, preferred aspartic acid and tyrosine be increased in 308% and 698%, preferred 698% and 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 161st and/or 168 row) is enhanced, give each fine chemicals, preferred Serine and tyrosine be increased in 30% and 698%, preferred 698% and 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 134 and/or 161 row) is enhanced, give each fine chemicals, preferred L-Ala and aspartic acid and tyrosine be increased in 47% and 698%, preferred 308% and 698%, more preferably 698% and 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 134th and/or 161 and/or 168 row) is enhanced, give each fine chemicals, preferred aspartic acid Serine and tyrosine be increased in 30% and 698%, preferred 308% and 698%, more preferably 698% and 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 161 and/or 168 row) is enhanced, give each fine chemicals, preferred L-Ala and Serine and tyrosine be increased in 30% and 698%, preferred 47% and 698%, more preferably 698% and 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 134 and/or 161 and/or 168 row) is enhanced, give each fine chemicals, preferred L-Ala and aspartic acid and Serine and tyrosine be increased in 30% and 698%, preferred 47% and 698%, more preferably 308% and 698% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL072C or its homologue, for example chromosomal DNA duplicates the subunit of required GINS complex body, under the situation that the activity of the possible membranin YJL072c superfamily of preferably saccharomyces cerevisiae (shown in Table I the 5th or 7 row, the 145th row) is enhanced, give each fine chemicals, preferred phenylalanine be increased in 25% and 204% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YKR057W or its homologue, for example participate in the biological ribosomal protein that is similar to the S21 ribosomal protein that takes place and translate of rrna, under the situation that the activity of preferred rat ribosomal protein 21 superfamilies (shown in Table I the 5th or 7 row, the 160th row) is enhanced, give each fine chemicals, preferred Serine be increased in 24% and 170% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YLL009C or its homologue, for example under the activity situation about being enhanced of cytochrome c oxidase copper chaperone (shown in Table I the 5th or 7 row, the 159th row), give each fine chemicals, preferred Serine be increased in 27% and 84% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YLL013C or its homologue, for example under the situation that the activity of PUF protein families (it is from the member pumilio that found and Fbf name) (shown in Table I the 5th or 7 row, the 140th row) is enhanced, give each fine chemicals, preferred citrulline be increased in 30% and 44% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YLR082C or its homologue, for example under the activity situation about being enhanced of the invalid lethal arrestin of Rad53 (shown in Table I the 5th or 7 row, the 158th row), give each fine chemicals, preferred Serine be increased in 22% and 61% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOL123W or its homologue, for example participate in premessenger RNA 3 '-shearing and the poly-adenosine factor CF I composition of end processing, under the situation that the activity of preferred heterogeneity ribonucleoprotein HRP1-yeast (yeast saccharomyces cerevisiae) superfamily (shown in Table I the 5th or 7 row, the 141st row) is enhanced, give each fine chemicals, preferred glycine be increased in 35% and 105% or more between.

In one embodiment; at yeast saccharomyces cerevisiae protein YOR245C or its homologue; acetyl-CoA for example: Diacrylglycerol acyl transferase; under the situation that the activity of preferred Caenorhabditis elegans putative protein matter K07B1.4 superfamily (shown in Table I the 5th or 7 row, the 139th row) is enhanced, give each fine chemicals, preferred citrulline be increased in 47% and 69% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR261C or its homologue, for example proteasome is regulated the particle subunit, under the situation that the activity of preferred mov-34 protein superfamily (shown in Table I the 5th or 7 row, the 157th row) is enhanced, give each fine chemicals, preferred Serine be increased in 28% and 40% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR350C or its homologue, for example characterize yet but appear as under the situation that the activity of yeast saccharomyces cerevisiae MNE1 protein superfamily member (shown in Table I the 5th or 7 row, the 167th row) is enhanced, give each fine chemicals, preferred tyrosine be increased in 67% and 177% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPR138C or its homologue, subunit NH4+ transporter for example, under the situation that the activity of preferred ammonium transporter and/or ammonium transporter nrgA superfamily (shown in Table I the 5th or 7 row, the 144th row) is enhanced, give each fine chemicals, preferred phenylalanine be increased in 49% and 93% or more between.

In one embodiment, at e. coli k12 protein b0695 or its homologue, the sensibility histidine kinase in the two-pack regulator control system for example, under the situation that the activity of preferred sensibility histidine kinase homology superfamily (shown in Table I the 5th or 7 row, the 156th row) is enhanced, give each fine chemicals, preferred phenylalanine be increased in 36% and 74% or more between.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example succinic thiokinase operon transcriptional and/or acyl are replied regulatory gene, under the situation that the activity of preferred transcriptional GntR superfamily (shown in Table I the 5th or 7 row, the 138th row) is enhanced, give each fine chemicals, preferred aspartic acid be increased in 47% and 268% or more between.

In one embodiment, at e. coli k12 protein b1708 or its homologue, lipoprotein for example, under the situation that the activity of preferred conservative putative protein matter HI1314 superfamily (shown in Table I the 5th or 7 row, the 154th row) is enhanced, give each fine chemicals, preferred phenylalanine be increased in 48% and 237% or more between.

In one embodiment, at e. coli k12 protein b1827 or its homologue, for example have the transcription repressor (IclR family) of DNA in conjunction with wing spirane structure territory, under the situation that the activity of preferred acetate operon repressor superfamily (shown in Table I the 5th or 7 row, the 153rd row) is enhanced, give each fine chemicals, preferred phenylalanine be increased in 40% and 293% or more between.

In one embodiment, at e. coli k12 protein b1827 or its homologue, for example have the transcription repressor (IclR family) of DNA in conjunction with wing spirane structure territory, under the situation that the activity of preferred acetate operon repressor superfamily (shown in Table I the 5th or 7 row, the 172nd row) is enhanced, give each fine chemicals, preferred tyrosine be increased in 40% and 228% or more between.

In one embodiment, at e. coli k12 protein b1827 or its homologue, for example have the transcription repressor (IclR family) of DNA in conjunction with wing spirane structure territory, under the situation that the activity of preferred acetate operon repressor superfamily (shown in Table I the 5th or 7 row, the 153rd and/or 172 row) is enhanced, give each fine chemicals, preferred phenylalanine and tyrosine be increased in 40% and 293%, preferred 40% and 293% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, the heat shock protein that for example has protease activity, under the situation that the activity of preferred heat shock protein htpX superfamily (shown in Table I the 5th or 7 row, the 166th row) is enhanced, give each fine chemicals, preferred Serine be increased in 24% and 166% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, the heat shock protein that for example has protease activity, under the situation that the activity of preferred heat shock protein htpX superfamily (shown in Table I the 5th or 7 row, the 171st row) is enhanced, give each fine chemicals, preferred tyrosine be increased in 56% and 741% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, the heat shock protein that for example has protease activity, under the situation that the activity of preferred heat shock protein htpX superfamily (shown in Table I the 5th or 7 row, the 166th and/or 171 row) is enhanced, give each fine chemicals, preferred Serine and tyrosine be increased in 24% and 741%, preferred 56% and 741% or more between.

In one embodiment, at e. coli k12 protein b2095 or its homologue, tagatose-6-phosphokinase for example, under the situation that the activity of the possible tagatose 6-phosphokinase gatZ superfamily of preferred Escherichia (shown in Table I the 5th or 7 row, the 133rd row) is enhanced, give each fine chemicals, preferred L-Ala be increased in 22% and 33% or more between.

In one embodiment, at e. coli k12 protein b3008 or its homologue, PLP dependency cystathionine beta-lyase (beta-Cystathionase) for example, under the situation that the activity of preferred O-succinyl homoserine (sulfydryl)-lyase superfamily (shown in Table I the 5th or 7 row, the 132nd row) is enhanced, give each fine chemicals, preferred L-Ala be increased in 27% and 71% or more between.

In one embodiment, at e. coli k12 protein b3256 or its homologue, for example acetyl CoA carboxylase and/or biotin carboxylase enzyme subunit, under the situation that the activity of preferred biotin carboxylase superfamily (shown in Table I the 5th or 7 row, the 151st row) is enhanced, give each fine chemicals, preferred phenylalanine be increased in 44% and 50% or more between.

In one embodiment, at e. coli k12 protein b1697 or its homologue, for example has ETFP adenine nucleotide-in conjunction with the electron transfer flavoprotein subunit in spline structure territory, preferred electron shifts under the situation that the activity of flavoprotein β chain superfamily (shown in Table I the 5th or 7 row, the 155th row) is enhanced, give each fine chemicals, preferred phenylalanine be increased in 30% and 188% or more between.

In one embodiment, at e. coli k12 protein b1886 or its homologue, for example methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter acceptor, under the activity situation about being enhanced of preferable methyl acceptor chemotactic protein matter superfamily (shown in Table I the 5th or 7 row, the 152nd row), give each fine chemicals, preferred phenylalanine be increased in 33% and 196% or more between.

In one embodiment, at e. coli k12 protein b1886 or its homologue, for example methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter acceptor, under the activity situation about being enhanced of preferable methyl acceptor chemotactic protein matter superfamily (shown in Table I the 5th or 7 row, the 165th row), give each fine chemicals, preferred Serine be increased in 25% and 111% or more between.

In one embodiment, at e. coli k12 protein b1886 or its homologue, for example methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter acceptor, under the activity situation about being enhanced of preferable methyl acceptor chemotactic protein matter superfamily (shown in Table I the 5th or 7 row, the 152nd and/or 165 row), give each fine chemicals, preferred phenylalanine and Serine be increased in 25% and 196%, preferred 33% and 196% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, trehalose-6-phosphate synthase for example, preferred schizosaccharomyces pombe α, under the situation that the activity of α-trehalose-6-phosphate synthase (form UdP) superfamily (shown in Table I the 5th or 7 row, the 127th row) is enhanced, give each fine chemicals, preferred 5-oxyproline be increased in 34% and 150% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, trehalose-6-phosphate synthase for example, preferred schizosaccharomyces pombe α, under the situation that the activity of α-trehalose-6-phosphate synthase (form UdP) superfamily (shown in Table I the 5th or 7 row, the 137th row) is enhanced, give each fine chemicals, preferred aspartic acid be increased in 90% and 255% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, trehalose-6-phosphate synthase for example, preferred schizosaccharomyces pombe α, under the situation that the activity of α-trehalose-6-phosphate synthase (form UdP) superfamily (shown in Table I the 5th or 7 row, the 127th and/or 137 row) is enhanced, give each fine chemicals, preferred 5-oxyproline and aspartic acid be increased in 34% and 255%, preferred 90% and 255% or more between.

In one embodiment, at e. coli k12 protein b3462 or its homologue, conformity membrane cell division protein for example, under the activity situation about being enhanced of preferred cell split protein ftsX superfamily (shown in Table I the 5th or 7 row, the 150th row), give each fine chemicals, preferred phenylalanine be increased in 26% and 140% or more between.

In one embodiment, at e. coli k12 protein b3462 or its homologue, conformity membrane cell division protein for example, under the activity situation about being enhanced of preferred cell split protein ftsX superfamily (shown in Table I the 5th or 7 row, the 164th row), give each fine chemicals, preferred Serine be increased in 26% and 64% or more between.

In one embodiment, at e. coli k12 protein b3462 or its homologue, conformity membrane cell division protein for example, under the activity situation about being enhanced of preferred cell split protein ftsX superfamily (shown in Table I the 5th or 7 row, the 127th and/or 137 row), give each fine chemicals, preferred phenylalanine and Serine be increased in 26% and 140%, preferred 26% and 140% or more between.

At e. coli k12 protein b0057 or its homologue, for example have such as in [0022.0.14.14] the active protein of definition (as Table II the 5th or 7 row, shown in the 517th and 530 and 546 row) activity situation about being enhanced under, preferably, give fine chemicals in one embodiment, preferred citrulline is increased between 33% and 119%, preferred glycine is increased between 43% and 67%, preferred Serine is increased between 32% and 61%, preferred citrulline and Serine are increased between 32% and 119%, preferred citrulline and glycine are increased between 33% and 119%, preferred glycine and Serine are increased between 32% and 67%, preferred citrulline and glycine and Serine be increased in 32% and 119% or more between.

At e. coli k12 protein b0161 or its homologue, for example the pericentral siphon serine protease is (as Table II the 5th or 7 row, shown in the 492nd and 505 and 537 row) activity situation about being enhanced under, preferably, give fine chemicals in one embodiment, preferred 5-oxyproline be increased in 45% and 123% or more between, preferred aspartic acid be increased in 57% and 116% or more between, preferred phenylalanine be increased in 37% and 668% or more between, preferred 5-oxyproline and aspartic acid be increased in 45% and 123% or more between, preferred 5-oxyproline and phenylalanine be increased in 37% and 668% or more between, preferred aspartic acid and phenylalanine be increased in 37% and 668% or more between, preferred 5-oxyproline and aspartic acid and phenylalanine be increased in 37% and 668% or more between.

At e. coli k12 protein b0236 or its homologue, as Table II the 5th or 7 row, the 497th the row shown in), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred L-Ala be increased in 22% and 41% or more between.

At e. coli k12 protein b0376 or its homologue, for example under the situation about being enhanced in conjunction with the activity of the protein β-Nei Xiananmei/D-alanine carboxypeptidase of penicillin (shown in Table II the 5th or 7 row, the 493rd row), preferably, give in one embodiment fine chemicals, preferred 5-oxyproline be increased in 41% and 100% or more between.

At e. coli k12 protein b0462 or its homologue (shown in Table II the 5th or 7 row, the 518th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 36% and 44% or more between.

At e. coli k12 protein b0486 or its homologue, for example under the activity situation about being enhanced of amino acid/amine transporter (APC family) (shown in Table II the 5th or 7 row, the 498th and 547 row), preferably, give in one embodiment that each fine chemicals, preferred L-Ala are increased in 44% and 52%, preferred Serine be increased in 27% and 49% between, preferred L-Ala and Serine be increased in 27% and 52% or more between.

At e. coli k12 protein b0577 or its homologue, for example under the activity situation about being enhanced of Jia Ding translocator (shown in Table II the 5th or 7 row, the 506th and 531 row), preferably, give in one embodiment fine chemicals, preferred glycine be increased in 36% and 50% between, preferred aspartic acid be increased in 56% and 65% between, preferred glycine and aspartic acid be increased in 36% and 65% or more between.

At e. coli k12 protein b0970 or its homologue, for example under the activity situation about being enhanced of glutamate receptor (shown in Table II the 5th or 7 row, the 494th and 555 row), preferably, in one embodiment, give each fine chemicals, preferred 5-oxyproline be increased in 37% and 203% between, preferred tyrosine be increased in 35% and 498% between, preferred 5-oxyproline and tyrosine be increased in 35% and 498% or more between.

At e. coli k12 protein b1228 or its homologue (shown in Table II the 5th or 7 row, the 538th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 35% and 44% or more between.

At e. coli k12 protein b1275 or its homologue (shown in Table II the 5th or 7 row, the 519th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 32% and 50% or more between.

In one embodiment, (be listed as at e. coli k12 protein b1343 or its homologue as Table II the 5th or 7, shown in the 495th and 499 and 507 row), under the situation that the activity of proteins that for example participates in rRNA processing and/or translate is enhanced, preferably under the situation that the activity of the ATP RNA-dependent helicase that is stimulated by 23S rRNA or its homologue is enhanced, preferably, give each fine chemicals, preferred 5-oxyproline is increased between 37% and 87%, preferred L-Ala is increased between 27% and 44%, preferred aspartic acid is increased between 76% and 136%, preferred L-Ala and 5-oxyproline are increased in 27% and 87%, preferred L-Ala and aspartic acid are increased between 27% and 136%, preferred 5-oxyproline and aspartic acid are increased between 37% and 136%, preferred L-Ala and 5-oxyproline and aspartic acid be increased in 27% and 136% or more between.

In one embodiment, at e. coli k12 protein b1360 or have the active protein of dna replication protein matter or its homologue of the supposition of being defined as, for example under the activity situation about being enhanced of transcriptional or its homologue (shown in Table II the 5th or 7 row, the 520th row), preferably, in one embodiment, give fine chemicals, preferred citrulline be increased in 34% and 72% or more between.

At e. coli k12 protein b1863 or its homologue (shown in Table II the 5th or 7 row, the 500th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred L-Ala be increased in 22% and 45% or more between.

At e. coli k12 protein b2023 or its homologue (shown in Table II the 5th or 7 row, the 508th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 56% and 81% or more between.

At e. coli k12 protein b2078 or its homologue (shown in Table II the 5th or 7 row, the 539th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 26% and 89% or more between.

At e. coli k12 protein b2239 or its homologue (shown in Table II the 5th or 7 row, the 521st row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 37% and 73% or more between.

In one embodiment, at e. coli k12 protein b2414 or its homologue (for example Table II the 5th or 7 row, shown in the 522nd and 532 and 548 and 540 row) activity situation about being enhanced under, for example under the situation that threonine dehydra(ta)se superfamily activity of proteins is enhanced, the preferred amino acid bio that participates in synthesizes, halfcystine-aromatic series biosynthesizing, halfcystine-die aromatischen Aminosaeuren degraded, nitrogen and sulphur utilization, the aspartate family biosynthesizing, the aspartate family amino acid degradation, sulfuric acid and the biosynthesizing of L-cysteine derivative, biosynthesizing from elementary amino acid whose secondary species, from glycine, the biosynthesizing of the secondary species of L-Serine and L-L-Ala, under the situation that pyridoxal phosphate bonded activity of proteins is enhanced, under the situation that the activity of preferred PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue is enhanced, preferably, give each fine chemicals, preferred citrulline is increased between 31% and 40%, preferred glycine is increased between 38% and 62%, preferred Serine is increased between 27% and 53%, preferred phenylalanine is increased between 27% and 197%, preferred citrulline and glycine are increased between 31% and 62%, preferred citrulline and Serine are increased between 27% and 53%, preferred citrulline and phenylalanine are increased between 27% and 197%, preferred glycine and Serine are increased between 27% and 62%, preferred glycine and phenylalanine are increased between 27% and 197%, preferred Serine and phenylalanine are increased between 27% and 197%, preferred citrulline and glycine and Serine are increased between 27% and 62%, preferred citrulline and glycine and phenylalanine are increased between 27% and 197%, preferred citrulline and Serine and phenylalanine are increased between 27% and 197%, preferred glycine and Serine and phenylalanine are increased between 27% and 197%, preferred citrulline and glycine and Serine and phenylalanine be increased in 27% and 197% or more between.

In one embodiment, at e. coli k12 protein b2426 or its homologue (shown in Table II the 5th or 7 row, the 523rd row), for example have under the situation that the activity of the oxydo-reductase of NAD (P) binding domains is enhanced, preferably, give each fine chemicals, preferred citrulline be increased in 32% and 41% or more between.

In one embodiment, at e. coli k12 protein b2489 or its homologue (Table II the 5th for example or 7 row, the 524th, 533, shown in 549 and 501 row), for example under the situation that the activity of hydrogenase Fe-S subunit is enhanced, preferably, give each fine chemicals, preferred citrulline is increased between 33% and 60%, preferred glycine is increased between 33% and 78%, preferred Serine is increased between 23% and 47%, preferred L-Ala is increased between 21% and 27%, preferred citrulline and glycine are increased between 33% and 78%, preferred citrulline and Serine are increased between 23% and 60%, preferred citrulline and L-Ala are increased between 21% and 60%, preferred glycine and Serine are increased between 23% and 78%, preferred glycine and L-Ala are increased between 21% and 78%, preferred Serine and L-Ala are increased between 21% and 47%, preferred citrulline and glycine and Serine are increased between 23% and 78%, preferred citrulline and glycine and L-Ala are increased between 21% and 78%, preferred citrulline and Serine and L-Ala are increased between 21% and 60%, preferred glycine and Serine and L-Ala are increased between 21% and 78%, preferred citrulline and glycine and Serine and L-Ala be increased in 21% and 78% or more between.

At e. coli k12 protein b2491 or its homologue (shown in Table II the 5th or 7 row, the 556th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred tyrosine be increased in 40% and 89% or more between.

At e. coli k12 protein b2507 or its homologue (shown in Table II the 5th or 7 row, the 509th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 49% and 120% or more between.

In one embodiment, at e. coli k12 protein b2553 or its homologue (for example shown in Table II the 5th or 7 row, the 534th row), for example under the situation that the activity of the adjusting albumen P-II of glutamine synthetase is enhanced, preferably, give each fine chemicals, preferred glycine be increased in 36% and 83% or more between.

In one embodiment, at e. coli k12 protein b2576 or its homologue (for example Table III the 5th or 7 row, shown in the 502nd and 535 and 510 and 541 row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, give each fine chemicals, preferred L-Ala is increased between 24% and 49%, preferred glycine is increased between 40% and 73%, preferred aspartic acid is increased between 52% and 103%, preferred phenylalanine is increased between 23% and 41%, preferred L-Ala and glycine are increased between 24% and 73%, preferred L-Ala and aspartic acid are increased between 24% and 103%, preferred L-Ala and phenylalanine are increased between 23% and 49%, preferred glycine and aspartic acid are increased between 40% and 103%, preferred glycine and phenylalanine are increased between 23% and 73%, preferred aspartic acid and phenylalanine are increased between 23% and 103%, preferred L-Ala and glycine and aspartic acid are increased between 24% and 103%, preferred L-Ala and glycine and phenylalanine are increased between 23% and 73%, preferred L-Ala and aspartic acid and phenylalanine are increased between 23% and 103%, preferred glycine and aspartic acid and phenylalanine are increased between 23% and 103%, preferred L-Ala and glycine and aspartic acid and phenylalanine be increased in 23% and 103% or more between.

In one embodiment, e. coli k12 protein b2664 or have the supposition of being defined as have transcription repressor (GntR family) active protein or its homologue of DNA in conjunction with wing spirane structure territory, for example under the activity situation about being enhanced of transcriptional (shown in Table III the 5th or 7 row, the 550th row), preferably, in one embodiment, give the fine chemicals Serine be increased in 25% and 231% or more between.

At e. coli k12 protein b2753 or its homologue (shown in Table II the 5th or 7 row, the 511st row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 56% and 154% or more between.

At e. coli k12 protein b2796 or its homologue (shown in Table II the 5th or 7 row, the 542nd row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 33% and 105% or more between.

At e. coli k12 protein b3064 or its homologue (shown in Table II the 5th or 7 row, the 551st row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred Serine be increased in 39% and 84% or more between.

Under the situation that the activity of e. coli k12 protein b3116 or its homologue (shown in Table II the 5th or 7 row, the 512nd and 552 row) is enhanced, for example under the situation that the activity of the L-of anaerobic induction Threonine/L-Serine permease (HAAAP family) is enhanced, preferably, give each fine chemicals, preferred Serine be increased in 35% and 70% between, preferred aspartic acid be increased in 50% and 130% between, preferred Serine and aspartic acid be increased in 35% and 130% or more between.

Under the situation that the activity of e. coli k12 protein b3160 or its homologue (shown in Table II the 5th or 7 row, the 525th and 553 row) is enhanced, for example have under the situation that the activity of the monooxygenase of luciferase sample atpase activity is enhanced, preferably, give each fine chemicals, preferred citrulline be increased in 34% and 48% between, preferred Serine be increased in 31% and 60% between, preferred citrulline and Serine be increased in 31% and 60% or more between.

At e. coli k12 protein b3169 or its homologue, for example under the activity situation about being enhanced of Transcription Termination-antitermination factor (shown in Table II the 5th or 7 row, the 513rd row), preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 66% and 168% or more between.

At e. coli k12 protein b3172 or its homologue (shown in Table II the 5th or 7 row, the 496th and 514 row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 52% and 234% between, preferred 5-oxyproline be increased in 44% and 94% between, preferred aspartic acid and 5-oxyproline be increased in 44% and 234% or more between.

At e. coli k12 protein b3231 or its homologue, for example under the activity situation about being enhanced of 50S ribosomal subunit protein matter L13 (shown in Table II the 5th or 7 row, the 503rd and 554 row), preferably, give in one embodiment fine chemicals, preferred L-Ala be increased in 21% and 35% between, preferred Serine be increased in 23% and 49% between, preferred L-Ala and Serine be increased in 21% and 49% or more between.

At e. coli k12 protein b3241 or its homologue (shown in Table II the 5th or 7 row, the 526th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 38% and 70% or more between.

At e. coli k12 protein b3767 or its homologue (shown in Table II the 5th or 7 row, the 504th and 536 row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred L-Ala be increased in 24% and 70% between, preferred homoserine be increased in 24% and 71% between, preferred L-Ala and homoserine be increased in 24% and 71% or more between.

At e. coli k12 protein b3919 or its homologue, for example under the activity situation about being enhanced of triosephosphate isomerase (shown in Table II the 5th or 7 row, the 543rd row), preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 30% and 188% or more between.

At e. coli k12 protein b3926 or its homologue (shown in Table II the 5th or 7 row, the 527th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 70% and 94% or more between.

At e. coli k12 protein b3938 or its homologue (shown in Table II the 5th or 7 row, the 544th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 34% and 40% or more between.

At e. coli k12 protein b3983 or its homologue, intestinal bacteria ribosomal protein L11 superfamily for example, preferably have under the activity situation about being enhanced of the active protein of t50S ribosomal subunit protein matter L12 (shown in Table II the 5th or 7 row, the 545th and 557 row), give each fine chemicals, preferred phenylalanine be increased in 37% and 266% between, preferred tyrosine be increased in 44% and 357% between, preferred phenylalanine and tyrosine be increased in 37% and 357% or more between.

At e. coli k12 protein b4129 or its homologue, for example Methionin tRNA synthetic enzyme is for example shown in Table II the 5th or 7 row, the 515th row) activity situation about being enhanced under, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 53% and 141% or more between.

At e. coli k12 protein b4214 or its homologue (shown in Table II the 5th or 7 row, the 528th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 32% and 102% or more between.

At e. coli k12 protein b4269 or its homologue (shown in Table II the 5th or 7 row, the 529th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 31% and 63% or more between.

At e. coli k12 protein b4346 or its homologue, for example under the activity situation about being enhanced of 5-methylcytosine specificity restriction enzyme McrBC composition (shown in Table II the 5th or 7 row, the 516th row), preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 63% and 94% or more between.

[0046.0.14.14] in one embodiment, yeast saccharomyces cerevisiae protein YAL049C or its homologue, for example the activity of YAL049C protein (shown in Table I the 5th or 7 row, the 149th row) is given each fine chemicals and other amino acid or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YBL015W or its homologue, the glycoprotein and/or the acetyl-CoA lytic enzyme that for example contain seminose, the activity of preferred acetyl-CoA lytic enzyme superfamily (shown in Table I the 5th or 7 row, the 131st row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YDL127W or its homologue, for example the activity of G1/S-specific cell cyclin PCL2 (cyclin HCS26 homologue) (shown in Table I the 5th or 7 row, the 126th row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YEL045C or its homologue, for example the activity of YEL045C protein (shown in Table I the 5th or 7 row, the 163rd row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YEL046C or its homologue, the activity of for example low specificity L-threonine aldolase (shown in Table I the 5th or 7 row, the 143rd row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YER152C or its homologue, the activity that for example is similar to tyrosine aminotransferase (shown in Table I the 5th or 7 row, the 162nd row) is given each fine chemicals and the other compound with amino acid active or its precursor to be increased.

In one embodiment, yeast saccharomyces cerevisiae protein YER173W or its homologue, the activity that for example participates in dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table I the 5th or 7 row, the 130th and/or 136 and/or 148 row) is given each fine chemicals and the other compound with amino acid active or its precursor to be increased.

In one embodiment, yeast saccharomyces cerevisiae protein YFL050C or its homologue, for example divalence and/or trivalent inorganic cation transporter, the activity of preferably magnesium and cobalt translocator superfamily (shown in Table I the 5th or 7 row, the 129th and/or 142 and/or 170 row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YGR101W or its homologue, the activity of for example flat rhombus proteolytic enzyme (shown in Table I the 5th or 7 row, the 147th row) is given each fine chemicals and the other compound with amino acid active or its precursor to be increased.

In one embodiment, yeast saccharomyces cerevisiae protein YGR104C or its homologue, the activity of for example flat rhombus proteolytic enzyme (shown in Table I the 5th or 7 row, the 135th row) is given each fine chemicals and the other compound with amino acid active or its precursor to be increased.

In one embodiment, yeast saccharomyces cerevisiae protein YHR130C or its homologue, for example the activity of YHR130C protein (shown in Table I the 5th or 7 row, the 146th and/or 169 row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example S phase (DNA synthetic) activity initial or that finish necessary chromobindins (shown in Table I the 5th or 7 row, the 128th and/or 134 and/or 161 and/or 168 row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YJL072C or its homologue, for example chromosomal DNA duplicates the subunit of required GINS complex body, and the activity of the possible membranin YJL072c superfamily of preferably saccharomyces cerevisiae (shown in Table I the 5th or 7 row, the 145th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YKR057W or its homologue, for example participate in that rrna is biological to be taken place and the ribosomal protein that is similar to the S21 ribosomal protein of translation, the activity of preferred rat ribosomal protein 21 superfamilies (shown in Table I the 5th or 7 row, the 160th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YLL009C or its homologue, for example the activity of cytochrome c oxidase copper chaperone (shown in Table I the 5th or 7 row, the 159th row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YLL013C or its homologue, for example the activity of PUF protein families (it is from the member pumilio that found and Fbf name) (shown in Table I the 5th or 7 row, the 140th row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YLR082C or its homologue, for example the activity of the invalid lethal arrestin of Rad53 (shown in Table I the 5th or 7 row, the 158th row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YOL123W or its homologue, for example participate in premessenger RNA 3 '-shearing and the poly-adenosine factor CF I composition of end processing, the activity of preferred heterogeneity ribonucleoprotein HRP1-yeast (yeast saccharomyces cerevisiae) superfamily (shown in Table I the 5th or 7 row, the 141st row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment; yeast saccharomyces cerevisiae protein YOR245C or its homologue; acetyl-CoA for example: Diacrylglycerol acyl transferase, the activity of preferred Caenorhabditis elegans putative protein matter K07B1.4 superfamily (shown in Table I the 5th or 7 row, the 139th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YOR261C or its homologue, for example proteasome is regulated the particle subunit, and the activity of preferred mov-34 protein superfamily (shown in Table I the 5th or 7 row, the 157th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YOR350C or its homologue for example characterize yet but the activity that appears as yeast saccharomyces cerevisiae MNE1 protein superfamily member (shown in Table I the 5th or 7 row, the 167th row) is given each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YPR138C or its homologue, subunit NH4+ transporter for example, the activity of preferred ammonium transporter and/or ammonium transporter nrgA superfamily (shown in Table I the 5th or 7 row, the 144th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b0695 or its homologue, the sensibility histidine kinase in the two-pack regulator control system for example, the activity of preferred sensibility histidine kinase homology superfamily (shown in Table I the 5th or 7 row, the 156th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b0730 or its homologue, for example succinic thiokinase operon transcriptional and/or acyl are replied regulatory gene, and the activity of preferred transcriptional GntR superfamily (shown in Table I the 5th or 7 row, the 138th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b1708 or its homologue, lipoprotein for example, the activity of preferred conservative putative protein matter HI1314 superfamily (shown in Table I the 5th or 7 row, the 154th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b1827 or its homologue, for example have the transcription repressor (IclR family) of DNA in conjunction with wing spirane structure territory, the activity of preferred acetate operon repressor superfamily (shown in Table I the 5th or 7 row, the 153rd and/or 172 row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b1829 or its homologue, the heat shock protein that for example has protease activity, the activity of preferred heat shock protein htpX superfamily (shown in Table I the 5th or 7 row, the 166th and/or 171 row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b2095 or its homologue, tagatose-6-phosphokinase for example, the activity of the possible tagatose 6-phosphokinase gatZ superfamily of preferred Escherichia (shown in Table I the 5th or 7 row, the 133rd row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b3008 or its homologue, PLP dependency cystathionine beta-lyase (beta-Cystathionase) for example, the activity of preferred O-succinyl homoserine (sulfydryl)-lyase superfamily (shown in Table I the 5th or 7 row, the 132nd row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b3256 or its homologue, for example acetyl CoA carboxylase and/or biotin carboxylase enzyme subunit, the activity of preferred biotin carboxylase superfamily (shown in Table I the 5th or 7 row, the 151st row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b1697 or its homologue, for example have ETFP adenine nucleotide-in conjunction with the electron transfer flavoprotein subunit in spline structure territory, the activity that preferred electron shifts flavoprotein β chain superfamily (shown in Table I the 5th or 7 row, the 155th row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b1886 or its homologue, for example methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter acceptor, the activity of preferable methyl acceptor chemotactic protein matter superfamily (shown in Table I the 5th or 7 row, the 152nd and/or 165 row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b1896 or its homologue, trehalose-6-phosphate synthase for example, preferred schizosaccharomyces pombe α, the activity of α-trehalose-6-phosphate synthase (forming UdP) superfamily (shown in Table I the 5th or 7 row, the 127th and/or 137 row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

In one embodiment, e. coli k12 protein b3462 or its homologue, conformity membrane cell division protein for example, the activity of preferred cell split protein ftsX superfamily (shown in Table I the 5th or 7 row, the 150th and/or 164 row) gives each fine chemicals and the other compound with amino acid active or its precursor increases.

At e. coli k12 protein b0057 or its homologue, for example have such as in [0022.0.14.14] the active protein of definition (as Table II the 5th or 7 row, shown in the 517th and 530 and 546 row) activity situation about being enhanced under, preferably, give fine chemicals in one embodiment, preferred citrulline is increased between 33% and 119%, preferred glycine is increased between 43% and 67%, preferred Serine is increased between 32% and 61%, preferred citrulline and Serine are increased between 32% and 119%, preferred citrulline and glycine are increased between 33% and 119%, preferred glycine and Serine are increased between 32% and 67%, preferred citrulline and glycine and Serine be increased in 32% and 119% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b0161 or its homologue, for example the pericentral siphon serine protease is (as Table II the 5th or 7 row, shown in the 492nd and 505 and 537 row) activity situation about being enhanced under, preferably, give fine chemicals in one embodiment, preferred 5-oxyproline be increased in 45% and 123% or more between, preferred aspartic acid be increased in 57% and 116% or more between, preferred phenylalanine be increased in 37% and 668% or more between, preferred 5-oxyproline and aspartic acid be increased in 45% and 123% or more between, preferred 5-oxyproline and phenylalanine be increased in 37% and 668% or more between, preferred aspartic acid and phenylalanine be increased in 37% and 668% or more between, preferred 5-oxyproline and aspartic acid and phenylalanine be increased in 37% and 668% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b0236 or its homologue (shown in Table II the 5th or 7 row, the 497th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred L-Ala be increased in 22% and 41% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b0376 or its homologue, for example under the situation about being enhanced in conjunction with the activity of the protein β-Nei Xiananmei/D-alanine carboxypeptidase of penicillin (shown in Table II the 5th or 7 row, the 493rd row), preferably, give in one embodiment fine chemicals, preferred 5-oxyproline be increased in 41% and 100% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b0462 or its homologue (shown in Table II the 5th or 7 row, the 518th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 36% and 44% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b0486 or its homologue, for example under the activity situation about being enhanced of amino acid/amine transporter (APC family) (shown in Table II the 5th or 7 row, the 498th and 547 row), preferably, in one embodiment, give that each fine chemicals, preferred L-Ala are increased between 44% and 52%, preferred Serine is increased between 27% and 49%, preferred L-Ala and Serine be increased in 27% and 52% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b0577 or its homologue, for example under the activity situation about being enhanced of Jia Ding translocator (shown in Table II the 5th or 7 row, the 506th and 531 row), preferably, give in one embodiment that fine chemicals, preferred glycine are increased between 36% and 50%, preferred aspartic acid is increased between 56% and 65%, preferred glycine and aspartic acid be increased in 36% and 65% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b0970 or its homologue, for example under the activity situation about being enhanced of glutamate receptor (shown in Table II the 5th or 7 row, the 494th and 555 row), preferably, in one embodiment, give that each fine chemicals, preferred 5-oxyproline are increased between 37% and 203%, preferred tyrosine is increased between 35% and 498%, preferred 5-oxyproline and tyrosine be increased in 35% and 498% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b1228 or its homologue (shown in Table II the 5th or 7 row, the 538th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 35% and 44% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b1275 or its homologue (shown in Table II the 5th or 7 row, the 519th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 32% and 50% or more between and give other amino acid or its precursor increases.

In one embodiment, (be listed as at e. coli k12 protein b1343 or its homologue as Table II the 5th or 7, shown in the 495th and 499 and 507 row) activity situation about being enhanced under, under the situation that the activity of proteins that for example participates in rRNA processing and/or translate is enhanced, preferably under the situation that the activity of the ATP RNA-dependent helicase that is stimulated by 23S rRNA or its homologue is enhanced, preferably, give each fine chemicals, preferred 5-oxyproline is increased between 37% and 87%, preferred L-Ala is increased between 27% and 44%, preferred aspartic acid is increased between 76% and 136%, preferred L-Ala and 5-oxyproline are increased between 27% and 87%, preferred L-Ala and aspartic acid are increased between 27% and 136%, preferred 5-oxyproline and aspartic acid are increased between 37% and 136%, preferred L-Ala and 5-oxyproline and aspartic acid be increased in 27% and 136% or more between and give other amino acid or its precursor increases.

In one embodiment, at e. coli k12 protein b1360 or have the active protein of dna replication protein matter or its homologue of the supposition of being defined as, for example under the activity situation about being enhanced of transcriptional or its homologue (shown in Table II the 5th or 7 row, the 520th row), preferably, in one embodiment, give fine chemicals, preferred citrulline be increased in 34% and 72% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b1863 or its homologue (shown in Table II the 5th or 7 row, the 500th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred L-Ala be increased in 22% and 45% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b2023 or its homologue (shown in Table II the 5th or 7 row, the 508th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 56% and 81% or more between.

At e. coli k12 protein b2078 or its homologue (shown in Table II the 5th or 7 row, the 539th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 26% and 89% or more between.

At e. coli k12 protein b2239 or its homologue (shown in Table II the 5th or 7 row, the 521st row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 37% and 73% or more between and give other amino acid or its precursor increases.

In one embodiment, (be listed as at e. coli k12 protein b2414 or its homologue as Table II the 5th or 7, shown in the 522nd and 532 and 548 and 540 row) activity situation about being enhanced under, for example under the situation that threonine dehydra(ta)se superfamily activity of proteins is enhanced, the preferred amino acid bio that participates in synthesizes, halfcystine-aromatic series biosynthesizing, halfcystine-die aromatischen Aminosaeuren degraded, nitrogen and sulphur utilization, the aspartate family biosynthesizing, the aspartate family amino acid degradation, sulfuric acid and the biosynthesizing of L-cysteine derivative, biosynthesizing from elementary amino acid whose secondary species, from glycine, the biosynthesizing of the secondary species of L-Serine and L-L-Ala, under the situation that pyridoxal phosphate bonded activity of proteins is enhanced, under the situation that the activity of preferred PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit or its homologue is enhanced, preferably, give each fine chemicals, preferred citrulline is increased between 31% and 40%, preferred glycine is increased between 38% and 62%, preferred Serine is increased between 27% and 53%, preferred phenylalanine is increased between 27% and 197%, preferred citrulline and glycine are increased between 31% and 62%, preferred citrulline and Serine are increased between 27% and 53%, preferred citrulline and phenylalanine are increased between 27% and 197%, preferred glycine and Serine are increased between 27% and 62%, preferred glycine and phenylalanine are increased between 27% and 197%, preferred Serine and phenylalanine are increased between 27% and 197%, preferred citrulline and glycine and Serine are increased between 27% and 62%, preferred citrulline and glycine and phenylalanine are increased between 27% and 197%, preferred citrulline and Serine and phenylalanine are increased between 27% and 197%, preferred glycine and Serine and phenylalanine are increased between 27% and 197%, preferred citrulline and glycine and Serine and phenylalanine be increased in 27% and 197% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2426 or its homologue (shown in Table II the 5th or 7 row, the 523rd row) is enhanced, for example have under the situation that the activity of the oxydo-reductase of NAD (P) binding domains is enhanced, preferably, give each fine chemicals, preferred citrulline be increased in 32% and 41% or more between and give other amino acid or its precursor increases.

In one embodiment, (be listed as at e. coli k12 protein b2489 or its homologue as Table II the 5th or 7, the 524th, 533, shown in 549 and 501 row) activity situation about being enhanced under, for example under the situation that the activity of hydrogenase Fe-S subunit is enhanced, preferably, give each fine chemicals, preferred citrulline is increased between 33% and 60%, preferred glycine is increased between 33% and 78%, preferred Serine is increased between 23% and 47%, preferred L-Ala is increased between 21% and 27%, preferred citrulline and glycine are increased between 33% and 78%, preferred citrulline and Serine are increased between 23% and 60%, preferred citrulline and L-Ala are increased between 21% and 60%, preferred glycine and Serine are increased between 23% and 78%, preferred glycine and L-Ala are increased between 21% and 78%, preferred Serine and L-Ala are increased between 21% and 47%, preferred citrulline and glycine and Serine are increased between 23% and 78%, preferred citrulline and glycine and L-Ala are increased between 21% and 78%, preferred citrulline and Serine and L-Ala are increased between 21% and 60%, preferred glycine and Serine and L-Ala are increased between 21% and 78%, preferred citrulline and glycine and Serine and L-Ala be increased in 21% and 78% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b2491 or its homologue (shown in Table II the 5th or 7 row, the 556th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred tyrosine be increased in 40% and 89% or more between.

At e. coli k12 protein b2507 or its homologue (shown in Table II the 5th or 7 row, the 509th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 49% and 120% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2553 or its homologue (shown in Table II the 5th or 7 row, the 534th row) is enhanced, for example under the situation that the activity of the adjusting albumen P-II of glutamine synthetase is enhanced, preferably, give each fine chemicals, preferred glycine be increased in 36% and 83% or more between and give other amino acid or its precursor increases.

In one embodiment, (be listed as at e. coli k12 protein b2576 or its homologue as Table III the 5th or 7, shown in the 502nd and 535 and 510 and 541 row) activity situation about being enhanced under, for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, give each fine chemicals, preferred L-Ala is increased between 24% and 49%, preferred glycine is increased between 40% and 73%, preferred aspartic acid is increased between 52% and 103%, preferred phenylalanine is increased between 23% and 41%, preferred L-Ala and glycine are increased between 24% and 73%, preferred L-Ala and aspartic acid are increased between 24% and 103%, preferred L-Ala and phenylalanine are increased between 23% and 49%, preferred glycine and aspartic acid are increased between 40% and 103%, preferred glycine and phenylalanine are increased between 23% and 73%, preferred aspartic acid and phenylalanine are increased between 23% and 103%, preferred L-Ala and glycine and aspartic acid are increased between 24% and 103%, preferred L-Ala and glycine and phenylalanine are increased between 23% and 73%, preferred L-Ala and aspartic acid and phenylalanine are increased between 23% and 103%, preferred glycine and aspartic acid and phenylalanine are increased between 23% and 103%, preferred L-Ala and glycine and aspartic acid and phenylalanine be increased in 23% and 103% or more between.

In one embodiment, e. coli k12 protein b2664 or have the supposition of being defined as have transcription repressor (GntR family) active protein or its homologue of DNA in conjunction with wing spirane structure territory, for example under the activity situation about being enhanced of transcriptional (shown in Table III the 5th or 7 row, the 550th row), preferably, in one embodiment, give the fine chemicals Serine be increased in 25% and 231% or more between and give other amino acid or its precursor and increase.

At e. coli k12 protein b2753 or its homologue (shown in Table II the 5th or 7 row, the 511st row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 56% and 154% or more between.

At e. coli k12 protein b2796 or its homologue (shown in Table II the 5th or 7 row, the 542nd row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 33% and 105% or more between.

At e. coli k12 protein b3064 or its homologue (shown in Table II the 5th or 7 row, the 551st row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred Serine be increased in 39% and 84% or more between and give other amino acid or its precursor increases.

(go at e. coli k12 protein b3116 or its homologue as Table II the 5th or 7 row, the 512nd and 552, be enhanced, for example under the situation that the activity of the L-of active anaerobic induction Threonine/L-Serine permease (HAAAP family) is enhanced, preferably, give that each fine chemicals, preferred Serine are increased between 35% and 70%, preferred aspartic acid is increased between 50% and 130%, preferred Serine and aspartic acid be increased in 35% and 130% or more between and give other amino acid or its precursor increases.

Under the situation that the activity of e. coli k12 protein b3160 or its homologue (shown in Table II the 5th or 7 row, the 525th and 553 row) is enhanced, for example have under the situation that the activity of the monooxygenase of luciferase sample atpase activity is enhanced, preferably, give that each fine chemicals, preferred citrulline are increased between 34% and 48%, preferred Serine is increased between 31% and 60%, preferred citrulline and Serine be increased in 31% and 60% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3169 or its homologue, for example under the activity situation about being enhanced of Transcription Termination-antitermination factor (shown in Table II the 5th or 7 row, the 513rd row), preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 66% and 168% or more between.

At e. coli k12 protein b3172 or its homologue (shown in Table II the 5th or 7 row, the 496th and 514 row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment that fine chemicals, preferred aspartic acid are increased between 52% and 234%, preferred 5-oxyproline is increased between 44% and 94%, preferred aspartic acid and 5-oxyproline be increased in 44% and 234% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3231 or its homologue, for example under the activity situation about being enhanced of 50S ribosomal subunit protein matter L13 (shown in Table II the 5th or 7 row, the 503rd and 554 row), preferably, give in one embodiment that fine chemicals, preferred L-Ala are increased between 21% and 35%, preferred Serine is increased between 23% and 49%, preferred L-Ala and Serine be increased in 21% and 49% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3241 or its homologue (shown in Table II the 5th or 7 row, the 526th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 38% and 70% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3767 or its homologue (shown in Table II the 5th or 7 row, the 504th and 536 row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment that fine chemicals, preferred L-Ala are increased between 24% and 70%, preferred homoserine is increased between 24% and 71%, preferred L-Ala and homoserine be increased in 24% and 71% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3919 or its homologue, for example under the activity situation about being enhanced of triosephosphate isomerase (shown in Table II the 5th or 7 row, the 543rd row), preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 30% and 188% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3926 or its homologue (shown in Table II the 5th or 7 row, the 527th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 70% and 94% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3938 or its homologue (shown in Table II the 5th or 7 row, the 544th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred phenylalanine be increased in 34% and 40% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b3983 or its homologue, for example active intestinal bacteria ribosomal protein L11 superfamily, preferably have under the activity situation about being enhanced of the active protein of t50S ribosomal subunit protein matter L12 (shown in Table II the 5th or 7 row, the 545th and 557 row), give that each fine chemicals, preferred phenylalanine are increased between 37% and 266%, preferred tyrosine is increased between 44% and 357%, preferred phenylalanine and tyrosine be increased in 37% and 357% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b4129 or its homologue, for example under the activity situation about being enhanced of Methionin tRNA synthetic enzyme (shown in Table II the 5th or 7 row, the 515th row), preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 53% and 141% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b4214 or its homologue (shown in Table II the 5th or 7 row, the 528th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 32% and 102% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b4269 or its homologue (shown in Table II the 5th or 7 row, the 529th row), for example have such as in [0022.0.14.14] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred citrulline be increased in 31% and 63% or more between and give other amino acid or its precursor increases.

At e. coli k12 protein b4346 or its homologue, for example under the activity situation about being enhanced of 5-methylcytosine specificity restriction enzyme McrBC composition (shown in Table II the 5th or 7 row, the 516th row), preferably, give in one embodiment fine chemicals, preferred aspartic acid be increased in 63% and 94% or more between and give other amino acid or its precursor increases.

[0047.0.0.14] to [0048.0.0.14]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.14.14] has to give and improves fine chemicals 5-oxyproline quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 126-127 and/or 492-496 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 126-127 and/or 492-496 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 126-127 and/or 492-496 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals L-Ala quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 128-133 and/or 497-504 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 128-133 and/or 497-504 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 128-133 and/or 497-504 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals aspartic acid quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 134-138 and/or 505-516 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 134-138 and/or 505-516 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 134-138 and/or 505-516 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals citrulline quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 139-140 and/or 517-529 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 139-140 and/or 517-529 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 139-140 and/or 517-529 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals glycine quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 141-142 and/or 530-535 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 141-142 and/or 530-535 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 141-142 and/or 530-535 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals homoserine quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in the 143rd and/or 536 row, as Table II the 5th or 7 row, the structure of its function homologue described in polypeptide or the literary composition shown in the 143rd and/or 536 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 143rd and/or 536 row) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals phenylalanine quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 144-156 and/or 537-545 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 144-156 and/or 537-545 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 144-156 and/or 537-545 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals Serine quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 157-166 and/or 546-554 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 157-166 and/or 546-554 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 157-166 and/or 546-554 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

Have to give and improve fine chemicals tyrosine quantity or the active protein of level preferably has polypeptide described herein, particularly comprise as Table IV the 7th row, the polypeptide of sequence of consensus sequence shown in 167-172 and/or 555-557 are capable, as Table II the 5th or 7 row, the structure of its function homologue shown in 167-172 and/or 555-557 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 167-172 and/or 555-557 are capable or its function homologue as herein described) coding, and have the activity described in the literary composition.

[0050.0.14.14] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, the for example sylvite of each fine chemicals, ammonium salt or sodium salt, perhaps each amino acid salts acidulants or vitriol of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine.

[0051.0.0.14] to [0052.0.0.14]: see that [0051.0.0.0] is to [0052.0.0.0]

[0053.0.14.14] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention (for example has as Table II the 3rd row, for the 126-127 of 5-oxyproline and/or 492-496 is capable and/or for the 128-133 of L-Ala and/or 497-504 is capable and/or for the 134-138 of aspartic acid and/or 505-516 is capable and/or for the 139-140 of citrulline and/or 517-529 is capable and/or for the 141-142 of glycine and/or 530-535 is capable and/or for the 143rd and/or 536 row of homoserine and/or for the 144-156 of phenylalanine and/or 537-545 is capable and/or for the 157-166 of Serine and/or 546-554 is capable and/or for the 167-172 of tyrosine and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, for the 126-127 of 5-oxyproline and/or 492-496 is capable and/or for the 128-133 of L-Ala and/or 497-504 is capable and/or for the 134-138 of aspartic acid and/or 505-516 is capable and/or for the 139-140 of citrulline and/or 517-529 is capable and/or for the 141-142 of glycine and/or 530-535 is capable and/or for the 143rd and/or 536 row of homoserine and/or for the 144-156 of phenylalanine and/or 537-545 is capable and/or for the 157-166 of Serine and/or 546-554 is capable and/or for the 167-172 of tyrosine and/or 555-557 capable shown in) active polypeptide) express and increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention and (are for example had as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or the of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by the coded protein of nucleic acid molecule of the present invention, polypeptide perhaps of the present invention (for example has as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active polypeptide) express and improve;

(e) by add the activity that one or more external source inducible factors come stimulating protein in biological or its part, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active polypeptide) express and improve;

(f) transgenosis of expression coded protein, described protein is given the activity with each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, and described nucleic acid molecule encoding has the active of each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active polypeptide);

(h) by adding positive Expression element or removing negative Expression element and improve code book invention polypeptide and (for example have such as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active polypeptide) and the expression of endogenous gene, for example can use homologous recombination in promoter, to introduce positive controlling element (such as plant 35S enhancer) or remove straining element from control region. Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) (heat shock protein(HSP) for example of the present invention) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.14.14] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having according to Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its homologue (for example be respectively Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide) give each fine chemicals after active expression of polypeptides or the activity and increase.

[0055.0.0.14] to [0071.0.0.14]: see that [0055.0.0.0] is to [0071.0.0.0]

[0072.0.14.14] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has other amino acid or precursor separately except 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine.

[0073.0.14.14] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) (for example Table II the 5th or 7 is listed as to improve polypeptide of the present invention or its homologue, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, for example give biology (preferred microorganism, the non-human animal, plant or zooblast, plant or animal tissues or plant) in each fine chemicals improve;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and other optional free and/or bonded amino acid.

[0074.0.0.14] to [0084.0.0.14]: see that [0075.0.0.0] is to [0084.0.0.0]

[0085.0.14.14] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as be respectively Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as be respectively Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.14] to [0088.1.0.14]: see that [0086.0.0.0] is to [0088.1.0.0]

[0089.0.0.14] to [0097.0.0.14]: see that [0089.0.0.0] is to [0097.0.0.0]

[0098.0.14.14] in preferred embodiments, each fine chemicals (5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine) be produce according to the present invention and carry out isolating where necessary.The method according to this invention produce other amino acid and aminoacid mixture or with the mixture of other compounds be favourable.

[0099.0.0.14] to [0102.0.0.14]: see that [0099.0.0.0] is to [0102.0.0.0]

[0103.0.14.14] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) the preferred nucleic acid molecule of encoding mature form polypeptide at least, described polypeptide have as Table II the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has as Table III the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) polypeptide of coding comprise have as Table IV the 7th row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecules that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.0.14.] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide of sequence.

[00103.2.0.14.] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide of sequence.

[0104.0.14.14] in one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention not by Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide of sequence.

[0105.0.0.14] to [0107.0.0.14]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.14.14] method of the present invention and had Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleic acid molecule of sequence, from Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in aminoacid sequence derive or from containing Table IV the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the polypeptide deutero-nucleic acid molecule of consensus sequence, perhaps its coding has as Table I the 3rd, 5 or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the enzymic activity of polypeptide or bioactive polypeptide or for example give derivative or the homologue that each fine chemicals increases behind its expression or active the increasing.

[0109.0.0.14]: see [0109.0.0.0]

[0110.0.4.4] ( II5、126-127/492-496/128-133/497-504/134-138/505-516/139-140/517-529/141-142/530-535/143/536/144-156/537-545/157-166/546-554/167-172/555-557I5、126-127/492-496/128-133/497-504/134-138/505-516/139-140/517-529/141-142/530-535/143/536/144-156/537-545/157-166/546-554/167-172/555-557 ( II7、126-127/492-496/128-133/497-504/134-138/505-516/139-140/517-529/141-142/530-535/143/536/144-156/537-545/157-166/546-554/167-172/555-557 ) ) 。

[0111.0.0.14]: see [0111.0.0.0]

The nucleotide sequence form of the nucleic acid molecules that uses in [0112.0.14.14] the inventive method for separating; Its coding has such as Table I the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide active polypeptide or have such as Table II the 5th and 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the polypeptide of peptide sequence, and give 5-OxoPro and/or alanine and/or aspartic acid and/or citrulling and/or glycine and/or homoserine and/or phenylalanine and/or serine and/or tyrosine and increase.

[0113.0.0.14] to [0120.0.0.14]: see that [0113.0.0.0] is to [0120.0.0.0]

[0121.0.14.14] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in peptide sequence or its functional homologue as herein described the difference of one or more amino acid moleculars is arranged, described artificial sequence is preferably given aforementioned activity, and promptly giving each fine chemicals behind its active increasing increases.

[0122.0.0.14] to [0127.0.0.14]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.14.14] PCR amplification uses is (for example such as Table III the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 primer shown in capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence or from such as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the sequence sequence generation of deriving.

[0129.0.14.14] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in consensus sequence from described comparison.

[0130.0.14.14] can use degenerated primer to have the new protein fragment of aforementioned activity (give amino acid as comprise this segmental protein expression or activity in raising after, for example 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine increase) by pcr amplification then.

[0131.0.0.14] to [0138.0.0.14]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.14.14] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna is listed as with Table I the 5th or 7 under loose hybridization conditions, preferred Table I B, for the 126-127 of 5-oxyproline and/or 492-496 is capable and/or for the 128-133 of L-Ala and/or 497-504 is capable and/or for the 134-138 of aspartic acid and/or 505-516 is capable and/or for the 139-140 of citrulline and/or 517-529 is capable and/or for the 141-142 of glycine and/or 530-535 is capable and/or for the 143rd and/or 536 row of homoserine and/or for the 144-156 of phenylalanine and/or 537-545 is capable and/or for the 157-166 of Serine and/or 546-554 is capable and/or for the 167-172 of tyrosine and/or 555-557 capable shown in sequence hybridization, and the coding expression has each fine chemicals, the particularly active peptide of 5-oxyproline and/or L-Ala and/or aspartic acid and/or citrulline and/or glycine and/or homoserine and/or phenylalanine and/or Serine and/or tyrosine of increasing.

[0140.0.0.14] to [0146.0.0.14]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.14.14] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table I B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.14.14] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, preferred Table I B, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, nucleotide sequence or its portion homologous are at least about 30% shown in the preferred Table I B, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases arginine and/or L-glutamic acid and/or proline(Pro) and/or glutamine.

[0149.0.14.14] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table I B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in one of nucleotide sequence or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and protein as shown in Table II.

[00149.1.14.14] randomly, with Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table I B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 protein shown in capable is known activity or is used for these protein of note.

[0150.0.14.14] in addition, nucleic acid molecule of the present invention can only contain Table I the 5th or 7 row, preferred Table I B, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving arginine and/or L-glutamic acid and/or proline (Pro) and/or glutamine when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and described nucleotides sequence column region is listed as with Table I the 5th or 7 under stringent condition, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the sense strand of one of sequence, Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or, 75 continuous nucleotide hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention. is listed as with Table III the 7th, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the right PCR of primer will produce such as Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in fragment or its gene outcome of polynucleotide sequence.

[0151.0.0.14] sees [0151.0.0.0]

[0152.0.14.14] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and are listed as Table II the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising 5-oxyproline as be shown in the examples and/or L-Ala and/or aspartic acid and/or citrulline and/or glycine and/or homoserine and/or phenylalanine and/or Serine and/or tyrosine level.

[0153.0.14.14] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence; Described aminoacid sequence comprises and is listed as Table I the 5th or 7; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example; Have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue); Produce so that this protein or its part can participate in increasing each fine chemicals. is listed as such as Table II the 5th or 7 in one embodiment, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein or its part have for example Table II the 3rd row as herein described, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the activity of polypeptide.

[0154.0.14.14] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% also most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.14] to [0156.0.0.14]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.14.14] the present invention relate in addition owing to genetic code degeneration be different from Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide; Particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise such as Table IV the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in consensus sequence polypeptide or such as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleic acid molecules of polypeptide or its function homologue. Advantageously; Nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein; And described protein comprises or has in another embodiment as Table IV the 7th row; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 consensus sequence shown in capable or as Table II the 5th or 7 be listed as; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment; Nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein; Described full length protein with comprise as Table IV the 7th row; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 consensus sequence shown in capable or as Table II the 5th or 7 be listed as; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet; In a preferred embodiment; Nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable; Preferred Table I A the 5th or 7 row; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable.Preferably; Nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in nucleic acid molecule identical.

[0158.0.0.14] to [0160.0.0.14]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.14.14] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or, 30 Nucleotide. preferably, (for example comprising Table I the 5th or 7 is listed as with the nucleic acid molecules that comprises the nucleotide sequence of nucleic acid molecules of the present invention or nucleic acid molecules that the inventive method is used under stringent condition for it, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleic acid molecules of sequence) hybridization. This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.14] sees [0162.0.0.0]

[0163.0.14.14] preferably, under stringent condition with Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.14] sees [0164.0.0.0]

[0165.0.14.14] for example, can the sequence of nucleic acid molecules of the present invention or the employed nucleic acid molecules of the inventive method (for example as Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence) in produce the amino acid substitution that causes " nonessential " amino acid residue place the nucleotides replacement.

[0166.0.0.14] to [0167.0.0.14]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.14.14] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table II B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the contained sequence difference of sequence, but kept activity as herein described.

This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contains and is listed as Table II the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table II B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 aminoacid sequence shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.

Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table II B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table II B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or one of 167-172 and/or the 555-557 sequence shown in capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table II B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table II B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.14] to [0172.0.0.14]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.14.14] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:21744 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:21744 sequence before use.

[0174.0.0.14] sees [0174.0.0.0]

[0175.0.14.14] for example, the sequence that has 80% homology at protein level and SEQ ID NO:21745 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:21745 sequence.

[0176.0.14.14] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the functional equivalent that obtains of one of polypeptide be listed as according to of the present invention as Table II the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide have essentially identical character and discern.

[0177.0.14.14] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table I B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the functional equivalent that obtains of nucleotide sequence be listed as according to of the present invention as Table II the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in one of polypeptide have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable, preferred Table I B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the polypeptide of the essentially identical character of polypeptide.

[0178.0.0.14]: see [0178.0.0.0]

[0179.0.14.14] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in) in introduce the replacement of one or more Nucleotide; Add or disappearance; And thereby in coded protein; Introduce one or more amino acid and replace; Add or disappearance and produce coding as Table II the 5th or 7 is listed as; Capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are capable; preferred Table II B the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleic acid molecule of homologue of protein sequence. ( PCR ) I57、126-127/492-496/128-133/497-504/134-138/505-516/139-140/517-529/141-142/530-535/143/536/144-156/537-545/157-166/546-554/167-172/555-557。

[0180.0.0.14] to [0183.0.0.14]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.14.14] is employed to have as Table I, preferred Table I B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the nucleotide sequence homologous compound of sequence, perhaps come Table II the 5th or 7 row freely, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the homologous compound of nucleotide sequence of sequence also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.14.14] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I, preferred Table I B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I, preferred Table I B the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 arbitrary sequence shown in capable in other Nucleotide of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.14.14] also preferred the inventive method comprise as Table II, preferred Table II B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II, preferred Table II B the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence identical.

[0187.0.14.14] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecules of the present invention or this method comprise as Table II the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence polypeptide and contain and be less than 100 other nucleotides. In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II, preferred Table II B the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in encoding sequence identical.

[0188.0.14.14] still has the polypeptide (=protein) of giving the basic biology of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in and under the same conditions the polypeptide expressed activity compare and do not reduce substantially.In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence or by its homologue of forming.

[0189.0.14.14] is as Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the homologous compound of sequence, or deutero-such as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or the homologue of 167-172 and/or the 555-557 sequence shown in capable also refers to truncated sequence, cDNA, the single stranded DNA or the RNA of coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.14] to [0203.0.0.14]: see that [0190.0.0.0] is to [0203.0.0.0]

[0204.0.14.14] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II, preferred Table II B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given each fine chemicals in biological or its part, particularly 5-oxyproline (126-127 and/or 492-496 are capable) and/or L-Ala (128-133 and/or 497-504 are capable) and/or aspartic acid (134-138 and/or 505-516 are capable) and/or citrulline (139-140 and/or 517-529 are capable) and/or glycine (141-142 and/or 530-535 are capable) and/or homoserine (the 143rd and/or 536 row) and/or phenylalanine (144-156 and/or 537-545 are capable) and/or Serine (157-166 and/or 546-554 are capable) and/or tyrosine (167-172 and/or 555-557 are capable) quantity increase;

(b) comprise, preferably comprise being listed as of mature form at least as Table I the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 primer shown in capable or primer obtain amplification from cDNA library or genomic library nucleic acid molecule, and in biological or its part, give each fine chemicals, the particularly increase of 5-oxyproline (126-127 and/or 492-496 are capable) and/or L-Ala (128-133 and/or 497-504 are capable) and/or aspartic acid (134-138 and/or 505-516 are capable) and/or citrulline (139-140 and/or 517-529 are capable) and/or glycine (141-142 and/or 530-535 are capable) and/or homoserine (the 143rd and/or 536 row) and/or phenylalanine (144-156 and/or 537-545 are capable) and/or Serine (157-166 and/or 546-554 are capable) and/or tyrosine (167-172 and/or 555-557 are capable) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) polypeptide of coding contains just like Table IV the 5th or 7 row; 126-127／492-496／128-133／497-504／134-138／505-516／139-140／517-529／141-142／530-535／143／536／144-156／537-545／157-166／546-554／167-172／555-557,5- （ 126-127／492-496 ） ／ （ 128-133／497-504 ） ／ （ 134-138／505-516 ） ／ （ 139-140／517-529 ） ／ （ 141-142／530-535 ） ／ （ 143／536 ） ／ （ 144-156／537-545 ） ／ （ 157-166／546-554 ） ／ （ 167-172／555-557 ） ；

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, the nucleic acid molecule that increases of 5-oxyproline (126-127 and/or 492-496 are capable) and/or L-Ala (128-133 and/or 497-504 are capable) and/or aspartic acid (134-138 and/or 505-516 are capable) and/or citrulline (139-140 and/or 517-529 are capable) and/or glycine (141-142 and/or 530-535 are capable) and/or homoserine (the 143rd and/or 536 row) and/or phenylalanine (144-156 and/or 537-545 are capable) and/or Serine (157-166 and/or 546-554 are capable) and/or tyrosine (167-172 and/or 555-557 are capable) quantity particularly, described peptide coding such as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or the structural domain of 167-172 and/or the 555-557 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 nucleic acid molecule shown in capable or coding (optimized encoding is mature form at least) as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the 15nt at least of nucleic acid molecule of polypeptide, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence;

Thereby the nucleic acid molecule of preferred (a) to (l) is different from as Table I A the 5th or 7 row, 126-127 by one or more Nucleotide and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable.

In one embodiment, nucleic acid molecule is not by Table I A or IB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable is formed.

In one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.

In another embodiment, nucleic acid molecule is not encoded as Table II A or IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 peptide sequence shown in capable.

In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence at least 30%, 40%, 50% or 60% is identical and less than 100%, 99.999%, 99.99%, 99.9% or 99% is identical.

In other embodiments, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in peptide sequence.

Therefore, in one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in nucleic acid molecule have at least one or more residues different.

Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide have the different polypeptide of one or more amino acid at least.

In another embodiment, Table I A or IB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in nucleic acid molecules do not encode as Table II A or IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the protein of sequence.

Therefore, in one embodiment, by nucleotide sequence (a)-(l) encoded protein matter not by Table II A or IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence form.

In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein sequence at least 30%, 40%, 50% or 60% identical and with Table I A or IIB the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% is identical.

[0205.0.0.14] to [0226.0.0.14]: see that [0205.0.0.0] is to [0226.0.0.0]

[0227.0.14.14] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the capable sequence or derivatives thereof of mentioning of 555-557, can be advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one amino acid biosynthetic pathway, for example amino acid precursor gene in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.0.14] to [0230.0.0.14]: see that [0228.0.0.0] is to [0230.0.0.0]

[00231.0.14.14] is in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded 5-oxyproline and/or L-Ala and/or aspartic acid and/or citrulline and/or glycine and/or homoserine and/or phenylalanine and/or Serine and/or tyrosine simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.14] to [0282.0.0.14]: see that [0232.0.0.0] is to [0282.0.0.0]

[0283.0.14.14] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in protein, perhaps as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the antibody of polypeptide, it can comprise above-mentioned sequence or by its polypeptide of forming by the standard technique utilization, and polypeptide for example of the present invention or its fragment produce.Monoclonal antibody preferably.

[0284.0.0.14] sees [0284.0.0.0]

[0285.0.14.14] the present invention relates to have such as Table II the 5th or 7 row in one embodiment, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence or by such as Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide or its function homologue of the coded sequence of nucleic acid molecules.

[0286.0.14.14] in an advantageous embodiment, improved in the inventive method contain just like Table IV the 7th row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in consensus sequence or by the activity of its polypeptide of forming.In another embodiment, the present invention relates to contain just like Table IV the 7th row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in consensus sequence or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the polypeptide of consensus sequence (each row).

[0287.0.0.14] to [0290.0.0.14]: see that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.14.14] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as Table II A or IIB the 5th or 7 row, 126-127 by one or more amino acid and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 sequence shown in capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence form.

[0292.0.0.14]: see [0292.0.0.0]

[0293.0.14.14] the present invention relates to give that each fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in other sequence of sequence phase region.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence form.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help Table I A or IB the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.14.14] in one embodiment, the present invention relates to have as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the polypeptide of activity of proteins, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or Table II B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence.

[0295.0.0.14] to [0297.0.0.14]: see that [0295.0.0.0] is to [0297.0.0.0]

The chemical of [0297.1.14.14] non-polypeptide of the present invention is for example not have Table II the 3rd, 5 or 7 row, the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the active polypeptide of polypeptide.

[0298.0.14.14] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as being listed as with Table II the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence, thereby protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the identical aminoacid sequence of sequence.

[0299.0.14.14] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 be listed as, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in one of nucleotide sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.

The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described. such as aforementioned, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the coded amino acid sequence of the nucleotide sequence of nucleotide sequence hybridization (preferred hybridize under stringent condition) or its homologue.

[0300.0.14.14] therefore, described in detail as this paper, polypeptide of the present invention is because natural variation or mutagenesis can be on aminoacid sequences and as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the sequence difference.Therefore, this polypeptide contains and is listed as Table II A or IIB the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the complete amino acid sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.14] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.14.14] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 aminoacid sequence shown in capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.14] sees [0303.0.0.0]

[0304.0.14.14] operation nucleic acid molecules of the present invention may cause generation basically have as Table II the 3rd row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide active and with as described in the protein that there are differences in sequence of wild-type protein. These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.14] to [0308.0.0.14]: see that [0305.0.0.0] is to [0308.0.0.0]

[0309.0.14.14] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 protein shown in capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 " the non-polypeptide of the present invention " or " other polypeptide " in capable is meant the aminoacid sequence with corresponding following proteins polypeptide, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 are proteinic and from the protein of identical or different biology shown in capable.In one embodiment, be not shown in Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or capable " the non-polypeptide of the present invention " or " other polypeptide " of 555-557 do not give that each fine chemicals increases in biology or its part.

[0310.0.0.14] to [0334.0.0.14]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.14.14] confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or the expression of 167-172 and/or the 555-557 nucleotide sequence shown in capable and/or its homologue especially effectively and favourable. Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates to when introducing in the biology, (or be derived from its cell in the time of in the preferred introduced plant, tissue, organ or seed), by reducing as Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the expression of nucleotide sequence and/or its homologue and the double stranded rna molecule (dsRNA molecule) that causes metabolic activity to change.Be used for reducing as Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.14] to [0342.0.0.14]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.14.14] is as describing, in order to cause effective reduction of expression, at dsRNA and as Table I the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.14] to [0361.0.0.14]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.14.14] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 polypeptide shown in capable, for example coding has protein such as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the polypeptide of polypeptide active) nucleic acid molecule.

Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Have as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II the 3rd row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide (for example have as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

[0363.0.0.14] to [0384.0.0.14]: see that [0363.0.0.0] is to [0384.0.0.0]

The fermented liquid that [0385.0.14.14] obtains with this kind approach, the fermented liquid that particularly contains 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine, dry matter content accounts for 7.5 to 25% of weight usually.At least when finishing, but particularly passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means that the concentration that can utilize sugar in the fermention medium during this period of time remains on or be reduced to 0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant) or these methods or partly from fermented liquid, shift out or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

Purifying amino acid of the present invention, the particularly additive method of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine are as US 6,551, pass through electrodialytic method described in 803.

[0386.0.0.14] to [0392.0.0.14]: see that [0386.0.0.0] is to [0392.0.0.0]

[0393.0.4.4] the present invention relates to identify and gives the method that the compound that becomes more meticulous in the cell produces the gene product that increases that it comprises following step in one embodiment:

(a) sample that will comprise candidate gene (be coded in and give the gene product that fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) evaluation and nucleic acid molecule of the present invention are particularly as Table I, preferred Table I B the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 nucleic acid molecule shown in capable is hybridized under lax stringent condition nucleic acid molecule, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.14] to [0399.0.0.14]: see that [0394.0.0.0] is to [0399.0.0.0]

[0399.1.14.14] it is contemplated that whether depend on such as Table II the 5th or 7 row, 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or the 555-557 polypeptide shown in capable or its homologue active or express by for example seeking the resistance of the medicine of blocking-up catalyst preparation and observing this effect; Each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high such as Table II the 5th or 7 row, capable and/or the 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the more almost identical phenotype of biology of activity of protein.

[0400.0.0.14] to [0423.0.0.14]: see that [0400.0.0.0] is to [0423.0.0.0]

[0424.0.14.14] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, agonist with the inventive method evaluation, the nucleic acid molecule of identifying with the inventive method can be used to produce fine chemicals or produce fine chemicals and one or more other amino acid, Threonine particularly, glutamine, L-glutamic acid, Xie Ansuan, l-asparagine, methionine(Met), halfcystine, leucine, proline(Pro), tryptophane, Xie Ansuan, Isoleucine and arginine.

Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.14] to [0460.0.0.14]: see that [0425.0.0.0] is to [0460.0.0.0]

[0461.0.14.14] embodiment 10: clone SEQ ID NO:18718 is used for expressing plant

[0462.0.0.14] sees [0462.0.0.0]

[0463.0.14.14] passes through pcr amplification SEQ ID NO:18718 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.14] to [0466.0.0.14]: see that [0464.0.0.0] is to [0466.0.0.0]

[0467.0.14.14] selects following primer sequence for genes of SEQ ID NO:18718:

I) forward primer (SEQ ID NO:18874)

atgaaataca?aggaaatcaa?tttcttc

Ii) reverse primer (SEQ ID NO:18875)

ctacatagtt?atgttattgg?tgatcg

[0468.0.0.14] to [0479.0.0.14]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.14.14] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:18718

[0481.0.0.14] to [0513.0.0.14]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.14.14] selects as another kind of, as (Plant Cell ﹠amp such as Geigenberger; Environ, 19,1996:43-55) the described amino acid that advantageously detects in the ethanol extraction that separates by HPLC.

The different plants of being analyzed the results are shown in following table:

??ORF	Metabolite	Method	??Min	??Max
					??YAL049C	Phenylalanine	??GC+LC	??1.30	??1.74
??YBL015W	L-Ala	??GC	??1.29	??3.04
					??YDL127W	The 5-oxyproline	??GC	??1.40	??2.27
??YEL045C	Serine	??GC	??1.23	??1.80

??YEL046C	Homoserine	??GC	??1.44	??2.77
					??YER152C	Serine	??GC	??1.23	??1.75
??YER173W	L-Ala	??GC	??1.21	??2.32
					??YER173W	Aspartic acid	??GC	??1.64	??1.93
??YER173W	Phenylalanine	??GC	??1.44	??1.99
					??YFL050C	L-Ala	??GC	??1.19	??2.04
??YFL050C	Glycine	??GC	??1.36	??1.74
					??YFL050C	Tyrosine	??GC	??1.42	??1.56
??YGR101W	Phenylalanine	??GC	??1.40	??1.92
					??YGR104C	Aspartic acid	??GC	??2.08	??2.26
??YHR130C	Phenylalanine	??LC	??1.77	??1.77
					??YHR130C	Tyrosine	??GC+LC	??1.51	??2.26
??YIL150C	L-Ala	??GC	??1.47	??4.28
					??YIL150C	Serine	??GC	??1.30	??4.22
??YIL150C	Aspartic acid	??GC	??4.08	??4.08
					??YIL150C	Tyrosine	??GC	??7.98	??7.98
??YJL072C	Phenylalanine	??GC	??1.25	??3.04
					??YKR057W	Serine	??GC	??1.24	??2.70
??YLL009C	Serine	??GC	??1.27	??1.84
					??YLL013C	Citrulline	??LC	??1.30	??1.44
??YLR082C	Serine	??GC	??1.22	??1.61
					??YOL123W	Glycine	??GC	??1.35	??2.05
??YOR245C	Citrulline	??LC	??1.47	??1.69
					??YOR261C	Serine	??GC	??1.28	??1.40
??YOR350C	Tyrosine	??GC	??1.67	??2.77
					??YPR138C	Phenylalanine	??GC+LC	??1.49	??1.93

??b0695	Phenylalanine	??GC+LC	??1.36	??1.74
					??b0730	Aspartic acid	??GC	??1.47	??3.68
??b1708	Phenylalanine	??GC	??1.48	??3.37
					??b1827	Phenylalanine	??GC	??1.40	??3.93
??b1827	Tyrosine	??GC	??1.40	??3.28
					??b1829	Serine	??GC	??1.24	??2.66
??b1829	Tyrosine	??GC+LC	??1.56	??8.41
					??b2095	L-Ala	??GC	??1.22	??1.33
??b3008	L-Ala	??GC	??1.27	??1.71
					??b3256	Phenylalanine	??GC	??1.44	??1.50
??b1697	Phenylalanine	??GC	??1.30	??2.88
					??b1886	Phenylalanine	??LC	??1.33	??2.96
??b1886	Serine	??GC	??1.25	??2.11
					??b1896	The 5-oxyproline	??GC	??1.34	??2.50
??b1896	Aspartic acid	??GC	??1.90	??3.55
					??b3462	Serine	??GC	??1.26	??1.64
??b3462	Phenylalanine	??GC+LC	??1.26	??2.40
					??b0057	Citrulline	??LC	??1.33	??2.19
??b0057	Glycine	??GC	??1.43	??1.67
					??b0057	Serine	??GC	??1.32	??1.61
??b0161	The 5-oxyproline	??GC	??1.45	??2.23
					??b0161	Aspartic acid	??GC	??1.57	??2.16
??b0161	Phenylalanine	??LC+GC	??1.37	??7.68
					??b0236	L-Ala	??GC	??1.22	??1.41
??b0376	The 5-oxyproline	??GC	??1.41	??2.00
					??b0462	Citrulline	??LC	??1.36	??1.44

??b0486	L-Ala	??GC	??1.44	??1.52
					??b0486	Serine	??GC	??1.27	??1.49
??b0577	Glycine	??GC	??1.36	??1.50
					??b0577	Aspartic acid	??GC	??1.56	??1.65
??b0970	The 5-oxyproline	??GC	??1.37	??3.03
					??b0970	Tyrosine	??GC	??1.35	??5.98
??b1228	Phenylalanine	??GC	??1.35	??1.44
					??b1275	Citrulline	??LC	??1.32	??1.50
??b1343	L-Ala	??GC	??1.27	??1.44
					??b1343	The 5-oxyproline	??GC	??1.37	??1.87
??b1343	Aspartic acid	??GC	??1.76	??2.36
					??b1360	Citrulline	??LC	??1.34	??1.72
??b1863	L-Ala	??GC	??1.22	??1.45
					??b2023	Aspartic acid	??GC	??1.56	??1.81
??b2078	Phenylalanine	??LC+GC	??1.26	??1.89
					??b2239	Citrulline	??LC	??1.37	??1.73
??b2414	Citrulline	??LC	??1.31	??1.40
					??b2414	Glycine	??GC	??1.38	??1.62
??b2414	Serine	??GC	??1.27	??1.53
					??b2414	Phenylalanine	??GC	??1.27	??2.97
??b2426	Citrulline	??LC	??1.32	??1.41
					??b2489	Citrulline	??LC	??1.33	??1.60
??b2489	L-Ala	??GC	??1.21	??1.27
					??b2489	Glycine	??GC	??1.33	??1.78
??b2489	Serine	??GC	??1.23	??1.47
					??b2491	Tyrosine	??GC	??1.40	??1.89

??b2507	Aspartic acid	??GC	??1.49	??2.20
					??b2553	Glycine	??GC	??1.36	??1.83
??b2576	L-Ala	??GC	??1.24	??1.49
					??b2576	Glycine	??GC	??1.40	??1.73
??b2576	Aspartic acid	??GC	??1.52	??2.03
					??b2576	Phenylalanine	??GC	??1.23	??1.41
??b2664	Serine	??GC	??1.25	??3.31
					??b2753	Aspartic acid	??GC	??1.56	??2.54
??b2796	Phenylalanine	??LC	??1.33	??2.05
					??b3064	Serine	??GC	??1.39	??1.84
??b3116	Serine	??GC	??1.35	??1.70
					??b3116	Aspartic acid	??GC	??1.50	??2.30
??b3160	Citrulline	??LC	??1.34	??1.48
					??b3160	Serine	??GC	??1.31	??1.60
??b3169	Aspartic acid	??GC	??1.66	??2.68
					??b3172	The 5-oxyproline	??GC	??1.44	??1.94
??b3172	Aspartic acid	??GC	??1.52	??3.34
					??b3231	L-Ala	??GC	??1.21	??1.35
??b3231	Serine	??GC	??1.23	??1.49
					??b3241	Citrulline	??LC	??1.38	??1.70
??b3767	L-Ala	??GC	??1.24	??1.70
					??b3767	Homoserine	??GC	??1.24	??1.71
??b3919	Phenylalanine	??GC	??1.30	??2.88
					??b3926	Citrulline	??LC	??1.70	??1.94
??b3938	Phenylalanine	??LC+GC	??1.34	??1.40
					??b3983	Phenylalanine	??LC+GC	??1.37	??3.66

??b3983	Tyrosine	??LC+GC	??1.44	??4.57
					??b4129	Aspartic acid	??GC	??1.53	??2.41
??b4214	Citrulline	??LC	??1.32	??2.02
					??b4269	Citrulline	??LC	??1.31	??1.63
??b4346	Aspartic acid	??GC	??1.63	??1.94

[0515.0.14.14] the 2nd row have shown the amino acid of being analyzed.The 4th row and the 5th row have shown the ratio of amino acid between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 6th row have been pointed out analytical procedure.

[0516.0.0.14] to [0552.0.0.14]: see that [0516.0.0.0] is to [0552.0.0.0]

[0552.1.14.14]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF title	Metabolite	??Min	??Max
				??YAL049C	Phenylalanine	??1.78	??1.86
??YIL150C	L-Ala	??1.63	??3.17
				??YIL150C	Serine	??1.25	??2.02
??YKR057W	Serine	??2.05	??12.64
				??YIL150C	Aspartic acid	??2.11	??3.56
??YIL150C	Tyrosine	??1.19	??2.09

Table 2 shows that phenylalanine and L-Ala and Serine and aspartic acid and tyrosine have increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YAL049C or YIL150C or YKR057W.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YAL049C or its homologue is enhanced in maize plant, preferably, give the fine chemicals phenylalanine and be increased between 78% and 86%.

In one embodiment, yeast saccharomyces cerevisiae protein YIL150C or its homologue in maize plant, for example the S phase (DNA synthetic) is initial or finish under the situation that the activity of necessary chromobindins or its homologue is enhanced, preferably, give the fine chemicals L-Ala be increased in 63% and 217% between and/or give the fine chemicals Serine be increased in 25% and 102% between and/or give the fine chemicals aspartic acid be increased in 111% and 256% between and/or give fine chemicals tyrosine and be increased between 19% and 109%.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YKR057W or the biological ribosomal protein that is similar to the S21 ribosomal protein that takes place and translate of participation rrna or its homologue is enhanced in maize plant, preferably, giving the fine chemicals Serine is increased between 105% and 1164%.

[0552.2.14.14]: see [0552.2.0.0]

[0553.0.14.14]

1. produce the method for 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine respectively, it comprises:

(a) improve in non-human being or its one or more parts or produce as Table II the 5th or 7 row, be respectively 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, produce respectively under the condition of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine and cultivate this biology.

2. produce the method for 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine respectively, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is as Table II the 5th or 7 row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide or its fragment, described nucleic acid molecule is given 5-oxyproline in biology or its part, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively;

B) contain just like Table I the 5th or 7 row, be respectively 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or the nucleic acid molecule of 167-172 and/or the 555-557 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases respectively of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases respectively of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by using as Table III the 7th row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give 5-oxyproline in biology or its part, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in consensus sequence polypeptide and give biology or its part in the 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine respectively.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) reclaim respectively and free or bonded 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine that optionally separating is produced by selected mutation biology or its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

C) nucleic acid molecule, coding is as Table II the 5th or 7 row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in polypeptide or its fragment, described nucleic acid molecule is given 5-oxyproline in biology or its part, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively;

D) contain just like Table I the 5th or 7 row, be respectively 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or the nucleic acid molecule of 167-172 and/or the 555-557 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases respectively of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases respectively of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by using as Table III the 7th row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give 5-oxyproline in biology or its part, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in consensus sequence polypeptide and give biology or its part in the 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, be respectively 126-127 and/or 492-496 is capable and/or 128-133 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, it produces by the method described in claim 13 or by the nucleic acid molecule encoding described in claim 6, this polypeptide is different from as Table II A the 5th or 7 row by one or more amino acid thus, be respectively the capable and/or 128-133 of 126-127 and/or 492-496 and/or 497-504 is capable and/or 134-138 and/or 505-516 is capable and/or 139-140 and/or 517-529 is capable and/or 141-142 and/or 530-535 is capable and/or the 143rd and/or 536 row and/or 144-156 and/or 537-545 is capable and/or 157-166 and/or 546-554 is capable and/or 167-172 and/or 555-557 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the agonist of screening polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity increase respectively, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity respectively increase coded by the nucleic acid molecule of claim 5 and candidate compound or comprise multiple compound contacts under the condition that allows this expression of polypeptides;

(b) measure in cell, tissue, plant or the microorganism, perhaps each level of 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) the 5-oxyproline by measuring, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, each level of Serine and/or tyrosine or expression of polypeptides level and the standard 5-oxyproline of when described candidate compound or the sample that comprises described multiple compound lack, measuring, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine expression level or expression of polypeptides level are separately relatively identified agonist or antagonist; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and give the method that 5-oxyproline in plant or the microorganism, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, the polypeptide that Serine and/or tyrosine quantity increase and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and it is coded and give 5-oxyproline in biology or its part to be provided at the nucleic acid molecule of described read-out system of permission and claim 6, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, response compound and described polypeptide bonded detectable signal under the condition of the expression of polypeptides that Serine and/or tyrosine quantity increase; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine in the cell produce the gene product of raising separately, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine increases respectively after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce respectively 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or the tyrosine level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare with wild-type, give 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine level raising separately in the host cell after it is expressed.

20. identify and to give the method that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine in the cell produce the gene product of raising separately, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine quantity in biology or its part or level improve separately after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce respectively 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or the tyrosine level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare with wild-type, give 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine level raising separately in the host cell after it is expressed.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or tyrosine increase separately after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying and can gives regulation and control 5-oxyproline, L-Ala, aspartic acid, citrulline, glycine, homoserine, phenylalanine, Serine and/or the tyrosine compound of level separately at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the nucleic acid molecule of claim 6, the polypeptide of claim 14, the nucleic acid construct of claim 7, claim 8 or 9 carrier, antagonist or agonist according to claim 17 evaluation, the antibody of claim 15, the plant of claim 16 or plant tissue, the results material of claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make anti-5-oxyproline and/or L-Ala and/or aspartic acid and/or citrulline and/or glycine and/or homoserine and/or phenylalanine and/or Serine and/or the tyrosine synthetic weedicide of suppressing of plant.

[0554.0.0.14] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.15] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.15] sees [0001.0.0.0]

[0002.0.15.15] oils and fat (at the glyceryl ester (triacylglycerol (TAG)) that chemically is lipid acid) have vital role in nutrition, but be used for non-food application more and more, for example lubricating oil, hydraulic efficiency oil, biofuel or be used for finite process compound, plasticizer, soap and washing agent (W.Lohs and the W.Friedt of coating, in Designer Oil Crops, D.J.Murphy writes (VCH, Weinheirn, Gerrnany, 1993)).The ideal oil of industrial application should comprise the lipid acid of specific type, and compares this lipid acid based on the coarse raw materials of petroleum products and can carry out sustainable supply with the emulative low price of tool.In addition, this type of lipid acid may have the reactive group except carboxyl, so that the target of other chemically modified to be provided.(

Deng, Science, the 268th volume, 681-686 page or leaf, 1995).

[0003.0.15.15] lipid acid and triacylglycerol are widely used in food and fodder industry, makeup and drug moiety.Whether be have free saturated or unsaturated fatty acids or the triacylglycerol that improve content saturated or unsaturated fatty acids, they are suitable for using the most widely if depending on them; Therefore, for example, with polyunsaturated fatty acid (=PUFA) be added into infant formulas to improve its nutritive value.Multiple lipid acid and triacylglycerol mainly are to obtain from microorganism such as fungi or from oil-produced vegetable comprises plant plankton and algae such as soybean, rape, Sunflower Receptacle and other, and lipid acid and triacylglycerol obtain with its triacylglycerol form usually in these sources.

[0004.0.15.15] basically, microorganism such as genus mortierella or oil-produced vegetable such as soybean, Semen Brassicae campestris or Sunflower Receptacle or algae such as Crytocodinium belong to or brown algae to belong to be the source of common fatty acids oils, at this moment fatty acids oils obtains with its triacylglycerol form usually.Alternatively, they can obtain from animal such as fish easily.Free fatty acids advantageously is prepared by the hydrolytic action of highly basic such as potassium hydroxide or sodium.

Other source of [0005.0.15.15] lipid acid is biological lipid member.Lipid is preferably phospholipid and/or glycolipid class, more preferably glyceryl phosphatide, galactolipid and/or sphingolipid.

[0006.0.15.15.] margaric acid is mentioned in early days for the first time at 19th-century.One of 1813, M.E.Chevreul discovery fat was made up of lipid acid, and called after these " margaric acids ", pearly gloss is glittering to make people remember that the Greek is used to describe pearl, margaron or margarite owing to it has.In the mid-19th century, W.H.Heintz shows that " margaric acid " that Chevreul finds is palmitinic acid and stearic uncertain mixture.

Now, term " margaric acid " is the common name of margaric acid (17:0), and it exists so that trace is natural.

Lipid acid with odd numbers of carbon atoms is present in the plant with the triacylglycerol form trace from Brazilian macadamia nut oil, Herba Dracocephali (Dracocephalum moldavica) oil, Poppy seed, palm, almond, Sunflower Receptacle or soybean.Margaric acid can obtain (1%) from the animal tallow separation, particularly obtains in the subcutaneus adipose tissue from the subcutaneous lipids of lamb.

Margaric acid can be the composition of satiety agent or sterilant.In addition, it is also as the composition in makeup, pharmaceutical preparation and feed and the food, for example as disclosed baking auxiliary among the US 20030143312 or according to US 20040097392 as the composition in the tensio-active agent.Margaric acid is mainly as the quantitative internal standard of lipid acid.In addition it can also to be used for the treatment of may be the sacred disease (US6,652,866) that is caused by yeast, fungi or the protein virus learned based on yeast or fungal pathogen, perhaps be used for anti-keratolytic-wound healing composition (US 5,641,814).Up to the present margaric acid is mainly produced with comparatively high amts by organic synthesis.

[0007.0.15.15.] 2-hydroxy fatty acid is a synthetic in the animal and plant tissue, and the main component of sphingolipid normally.The sphingolipid that contains the 2-hydroxy fatty acid is found in most biologies, comprises plant, yeast, worm, vertebrates and some bacterial species.

In plant, ceramide and sphingolipid are the lipid acid of 'alpha '-hydroxylation more than 95% fatty acid component.The carboxyl groups of ceramide tends to comprise long-chain (C16 to C26, but once in a while can be longer) odd and even number saturated or monoenoic fatty acid and relevant 2-D-hydroxy fatty acid, is not always the case at the plant and animal tissue.

Usually, the plant sphingolipid is made of the sphingosine skeleton, and wherein the sphingosine skeleton is that glycosylated and common acid amides is connected on the lipid acid of hydroxylated (very) long-chain, is called cerebroside.Cerebroside is the basic moiety of plasma membrane, participates in multiple victory physiological function, comprises signal transmission, exocytosis, protein grappling and vesicle protein matter transhipment (Matthes etc., Z.Naturforsch.57C, 843-852,2002).

In Mammals, 2-hydroxyl sphingolipid is present in brain in a large number, because most of myelin lipid galactosyl ceramide and sulfolipins comprise the 2-hydroxy fatty acid.In Mammals, the 2-hydroxy fatty acid that contains sphingolipid only is present in nerve and face tissue in a large number.In Mammals central authorities and peripheral nervous system, galactosyl ceramide and sulfolipins (the 3-sulfuric ester of galactosyl ceramide) are myelinic main lipid components.These sphingoglycolipids comprise at high proportion the 2-hydroxy fatty acid of (about 50%) and are the key components of myelin (4,5).

In yeast such as yeast saccharomyces cerevisiae, the overwhelming majority comprises the 2-hydroxy fatty acid.Compare with control cells, expressing human FA2H COS7 cell comprises the doubly higher levels of 2-hydroxyl of 3-20 ceramide (C16, C18, a C24 C24:1) and 2-hydroxy fatty acid (Alderson etc., J.Biol.Chem. the 279th volume, the 47th phase, the 48562-48568 page or leaf, 2004).

The 2-hydroxylation betides in the ceramide de novo synthesis process and is catalytic by lipid acid 2-hydroxylase (also being known as lipid acid α-hydroxylase).Non-free hydroxy fatty acid or hydroxy fatty acid CoA once reported in the past; But hydroxylated product (Hoshi etc., J.Biol.Chem.248,4123-4130,1973) occur as ceramide or cerebroside moiety usually.'alpha '-hydroxylation causes the direct hydroxylation of sphingolipid conjugated fatty acid.(Kayal etc., J.Biol.Chem. the 259th volume, the 6th phase, 3548-3553,1984).

Inflammatory reaction in the initial soft tissue of hydroxylation lipid acid is also regulated immune response.

2-oh group in the sphingolipid has far-reaching influence because its hydrogen bond forms ability lipid in film in forming.

α hydroxy-palmitic acid (hC 16:0) mainly is the formation unit of plant sphingolipid such as soybean glucosylceramide (GlcCer), and it is mainly by 4, and 8-sphingadiene skeleton and Alpha-hydroxy palmitinic acid are formed.The tumour that suppresses soybean GlcCer take place and mouse in genetic expression (Symolon etc., J.Nutr.2004 May; 134 (5): 1157-61).

Glucocerebroside (pinelloside) with strong antiseptic-germicide characteristic (at Gram-positive and negative bacteria and fungi) is described in Pinella ternata (Araceae) stem tuber, a kind of composition (Chen etc. of the decoction that uses in the tradition Chinese medicine, Phytochemistry 2003,64,903).Its structure shows and comprises glucose moiety and rare 4 of 2-hydroxy-palmitic acid, the 11-sphingadienine of being connected to.

It is 2-oxynervonic acid (2-OH-C24:1) that another kind constitutes unitary hydroxylation lipid acid as cerebroside.2-hydroxyl 15-tetracosenoic acid (oxynervonic acid) is the moiety (sphingoglycolipid mainly be found in nervous tissue and be found in the plant on a small quantity) of cerebroside ceramide part.The existence of 2-oxynervonic acid is the cerebronic feature of some cold-resistant cereal grass leaves (Sperling etc., BBA 1632,1-15,2003).

Hydroxylation lipid acid can be used for producing the method for fat or oils or be used to not have the lanolin cosmetic composition according to US 20040166130 according to US 20030054509.

Whether the oils that [0008.0.15.15] has unsaturated fatty acids or have a saturated fatty acid is preferred, and this depends on and is intended to purpose; Therefore, for example, the lipid that has unsaturated fatty acids, particularly polyunsaturated fatty acid in human nutrition is preferred, thereby has positive influence because they have positive influence to the cholesterol levels in the blood to cardiopathic possibility takes place.They are used for multiple nutrients food or medicine.In addition, PUFA is generally used for food, feed and cosmetic industry.Many unsaturated omega-3-fatty acids and/or ω-6-lipid acid is the integral part of animal-feed and human foods.Owing to the how unsaturated omega-3-fatty acid of human food prods's common component (they are main components of fish oil) should be added food to improve the nutritive value of food, therefore, for example polyunsaturated fatty acid such as DHA or EPA are added into as mentioned above in the formulated infant milk to improve its nutritive value.Real essential fatty acids linoleic and linolenic acid lipid acid have many active effects in human body, for example heart, artery and the skin for health has active effect.For example they can alleviate eczema, diabetic neuropathy or PMS and periodicity mastalgia.

Other how unsaturated ω-3-and/or ω among [0009.0.15.15.] animal-feed and the human food prods-6-lipid acid important component is Δ 7,10 hexadecadienoic acids (16:2 (n-6)) and Δ 7,10,13 hiragonic acids (16:3 (n-3)).Hexadecadienoic acid is the submember of some seeds and fish oil, and is the submember of plant leaf, and the precursor of hiragonic acid 16:3 (n-3) is the common composition of leaf lipid.Known this kind acid is present in photosynthetic leaf, as Arabidopis thaliana, rape leaf, pteridophyte lipid, ginkgo leaf, potato leaf, tomato leaf and spinach.This kind acid also is present in the leaf of Rhaphanus (Brassicaceae) plant, for example horseradish, wild cabbage, turnip, Chinese leaf mustard, Cauliflower and watercress.

More waiting in the plant, galactolipid comprises polyunsaturated fatty acid at high proportion, may be linolenic acid (18:3 (n-3)) up to 95% wherein.In this case, the single and the abundantest molecular species one of the digalactosyl diacylglycerol (DGDG) sn-1 and the sn-2 position that fix on glycerol backbone has 18:3.Plant for example pea (it has 18:3 as almost unique a kind of lipid acid in single semi-lactosi diacylglycerol (DGDG)) is called " 18:3 plant ".Other plant (for example Arabidopis thaliana) comprises the hiragonic acid (16:3 (n-3)) of ponderable amount in single semi-lactosi diacylglycerol (DGDG), be called as " 16:3 plant ".

As mentioned, polyunsaturated fatty acid also is used for cosmetic industry.Application US20030039672 disclose by topical application comprise that the combination treatment of the unsaturated C16 lipid acid (it is preferably hiragonic acid) that contains at least three two keys is aging, the beauty method of allergy, drying, scale, wrinkle and/or light injury skin.

[0010.0.15.15.] is because the positive characteristics of polyunsaturated fatty acid, spared no effort to prepare available in the past and participated in lipid acid or triacylglycerol synthetic gene, so that in the multiple biology of unsaturated fatty acid content, produce oils with change.

[0011.0.15.15] in the past few years used the recombinant DNA technology method by increasing each fatty acid biological synthetic gene and study its influence that lipid acid is produced and improve oil-contg in microorganism or the plant.For example, described Δ-9-desaturase in WO 91/13972, this enzyme participates in the synthetic of polyunsaturated fatty acid.In WO 93/11245 claimed Δ-15-desaturase and in WO 94/11516 claimed Δ-12-desaturase.Other desaturase has also been described, for example at EP-A-0550162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0794250, Stukey etc., J.Biol.Chem., 265,1990:20144-20149, Wada etc., Nature 347,1990:200-203 or Huang etc., Lipids, 34, among the 1999:649-659.Yet, up to now, because the multiple desaturase that exists with the membrane bound protein form all has very large difficulty on separation and sign, therefore these enzymes have only carried out some inadequate feature descriptions (McKeon etc., Method inEnzymol.71,1981:12141-12147 on biological chemistry, Wang etc., Plant Physiol.Biochem., 26,1988:777-792).Usually, characterize, by analyzing raw material and product enzymic activity is studied then by film is imported suitable biology in conjunction with desaturase.Effect of expressing about desaturase and the influence that polyunsaturated fatty acid is formed thereof can be noticed, as institute's descriptions up to now by expressing polyunsaturated fatty acid/lipid that desaturase and prolongation enzyme have only obtained lower aq.Therefore, need alternate and more effectively have the approach of higher output yield.

[0012.0.15.15] therefore still exists great demand new and more suitable gene, and described genes encoding participates in fatty acid biological synthetic enzyme and can produce some lipid acid and not form unnecessary byproduct in the particular industry scale.Selecting to be used for biosynthetic gene, above-mentioned two features are particularly importants.On the one hand, but still need to obtain the development of the highest intrinsic energy polyunsaturated fatty acid, on the other hand, produce byproduct in process of production as few as possible.

[0013.0.0.15] sees [0013.0.0.0]

[0014.0.15.15] therefore, in the first embodiment, the present invention relates to produce the method for fine chemicals, wherein fine chemicals is margaric acid (C17:0, margaric acid) and/or 2-hydroxy-palmitic acid (2-OH-C16:0, the Alpha-hydroxy palmitinic acid, C16:0OH) and/or 2-hydroxyl tetracosenoic acid (2-hydroxyl 15-tetracosenoic acid, oxynervonic acid, Alpha-hydroxy-tetracosenoic acid, C24:1 (n-9) OH, the 2-hydroxyl is along the 9-tetracosenoic acid, Δ 9 hydroxyls-tetracosenoic acid) and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids (C16:2 (n-6), along the suitable 10-hexadecadienoic acid of 7-)) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids (C16:3 (n-3), along the suitable 13-hiragonic acid of the suitable 10-of 7-, or contain margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 hiragonic acid)), 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) triglyceride level, lipid, oils or fat.Therefore, in the present invention, term " fine chemicals " is meant " triglyceride level of margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7; 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7; 10; 13 hiragonic acids) and/or the margaric acid that contains and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7; 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7; 10,13 hiragonic acids) as used herein, lipid, oils and/or fat ".In addition, term " fine chemicals " is meant and comprises margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 as used herein, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or contain margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) triacylglycerol, lipid, the fine chemicals of oils and/or fat.

[0015.0.15.15] in one embodiment, term " fine chemicals " meaning is meant margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or contain margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) triglyceride level, lipid, oils and/or fat.In whole specification sheets, term " fine chemicals " meaning is meant margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or contain margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) triglyceride level, lipid, oils and/or fat; Margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and salt, ester, thioesters or free form or be bonded to other compound (triacylglycerol for example, the glycolipid class, phosphatide etc.) margaric acid of form and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids).In preferred embodiments, term " fine chemicals " meaning is meant free form or its salt form or is bonded to glycerol backbone or is bonded to the glycerol-3-phosphate main chain or is bonded to sphingosine-phosphate backbone or is bonded to sphingosine-monose or oligosaccharides main chain or be bonded to margaric acid and/or 2-hydroxy-palmitic acid and/or the 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (the preferred Δ 7 of glycerine-3-monose or two sugar backbones, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids).Its triacylglycerol, lipid, oils, the fat or the lipid mixtures meaning are meant and contain any combination or free margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) any triacylglycerol, lipid, oils and/or fat, for example sphingolipid, phosphoglyceride, lipid, glyceryl phosphatide, galactolipid, glycolipid class such as sphingoglycolipid, phosphatide such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, the glycerine Serine, phosphatidylinositols or two glycerine Serine or monoacylglycerols, diacylglycerol or triacylglycerol or other fatty acid ester such as acetyl-CoA thioesters (it contains more saturated or unsaturated fatty acids in fatty acid molecule).

[0016.0.15.15] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YDR513W and/or YER156C and/or YLR255C and/or b1829 protein and/or YER173W protein and/or YFR042W protein and/or YOR317W protein and/or YGL205W and/or YIL150C and/or YKL132C and/or YOR344C and/or b3430 and/or b0161 and/or b0758 and/or b0057 and/or b1097 and/or b2078 and/or b3231 protein, perhaps have as Table II A or IIB the 3rd row, 174-185, peptide sequence shown in 558-563 is capable or have as Table I A or IB the 5th or 7 row, 174-185, the protein of the coded peptide sequence of nucleic acid molecule shown in 558-563 is capable; With

(b) in allowing described biology, produce fine chemicals, margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 for example, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) or comprise margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or under the condition of the fine chemicals of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) make biological growth.

Therefore, the present invention relates to produce the method for each fine chemicals, it comprises

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II A or IIB the 3rd row, 174-185,558-563 capable shown in activity of proteins, perhaps have by Table I A or IB the 5th or 7 row, 174-185,558-563 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce fine chemicals, lipid acid, particularly margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 for example, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or under the condition of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) make biological growth.

[0016.1.15.15] therefore, term " fine chemicals " looks like in one embodiment and is meant " margaric acid " or its homologue relevant with the capable listed full sequence of Table I-IV 174-177; And the meaning is meant and Table I-IV the 178th, 558 and relevant " 2-hydroxy-palmitic acid " or its homologue of the 559 listed full sequences of row in one embodiment; And the meaning is meant and Table I A or relevant " the 2-hydroxyl tetracosenoic acid " of IB the 179th listed full sequence of row in one embodiment; And the meaning is meant " hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) " relevant with the listed full sequence of Table I-IV the 180th row in one embodiment; And the meaning is meant " hiragonic acid (preferred Δ 7,10,13 hiragonic acids) " relevant with the capable listed full sequence of Table I-IV 181-185 in one embodiment; And the meaning is meant " tetracosenoic acid " or its homologue relevant with the capable listed full sequence of Table I-IV 560-563 in one embodiment.

Therefore, term " fine chemicals " can refer to " margaric acid " (" C17:0 " according to environment and context, " margaric acid ") and/or " 2-hydroxy-palmitic acid " (" 2-OH-C16:0 ", " Alpha-hydroxy palmitinic acid ", " C16:0OH ") and/or " 2-hydroxyl tetracosenoic acid " (" 2-hydroxyl 15-tetracosenoic acid ", " oxynervonic acid ", " Alpha-hydroxy-tetracosenoic acid ", " C24:1 (n-9) OH ", " the 2-hydroxyl is along the 9-tetracosenoic acid ", " Δ 9 hydroxyls-tetracosenoic acid ") and/or " hexadecadienoic acid " (preferred " Δ 7; 10 hexadecadienoic acids " (" C16:2 (n-6) ", " along the suitable 10-hexadecadienoic acid of 7-")) and/or " hiragonic acid " (preferred " Δ 7; 10,13 hiragonic acids " (" C16:3 (n-3) ", " along the suitable 13-hiragonic acid of the suitable 10-of 7-", " hiragonic acid ")).For the meaning of illustrating term " fine chemicals " is meant " margaric acid " (" C17:0 ", " margaric acid ") and/or " 2-hydroxy-palmitic acid " (" 2-OH-C16:0 ", " Alpha-hydroxy palmitinic acid ", " C16:0OH ") and/or " 2-hydroxyl tetracosenoic acid " (" 2-hydroxyl 15-tetracosenoic acid ", " oxynervonic acid ", " Alpha-hydroxy-tetracosenoic acid ", " C24:1 (n-9) OH ", " the 2-hydroxyl is along the 9-tetracosenoic acid ", " Δ 9 hydroxyls-tetracosenoic acid ") and/or " hexadecadienoic acid " (preferred " Δ 7; 10 hexadecadienoic acids " (" C16:2 (n-6) ", " along the suitable 10-hexadecadienoic acid of 7-")) and/or " hiragonic acid " (preferred " Δ 7; 10; 13 hiragonic acids " (" C16:3 (n-3) ", " along the suitable 13-hiragonic acid of the suitable 10-of 7-", " hiragonic acid ")) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid), term term " each fine chemicals " also can be used.

[0017.0.0.15] sees [0017.0.0.0]

[0018.0.0.15] sees [0018.0.0.0]

The method that [0019.0.15.15] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II A or IIB the 3rd row, 174-185, protein active shown in 558-563 is capable or by as Table I A or IB the 5th or 7 row, 174-185, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 558-563 was capable.

[0020.0.15.15] be surprisingly found out that, gives the margaric acid content increase of institute conversion plant as the yeast saccharomyces cerevisiae protein YDR513W shown in Table II the 3rd or 5 row, 174-176 are capable and/or YER156C and/or YLR255C and/or the e. coli k12 protein b1829 transgene expression in Arabidopis thaliana shown in Table I the 3rd or 5 row, the 177th row.Therefore, in one embodiment, described protein or be used to produce margaric acid as its homologue shown in Table II the 7th row, 174-177 are capable.

Be surprisingly found out that the yeast saccharomyces cerevisiae protein YER173W transgene expression in Arabidopis thaliana shown in Table II the 3rd or 5 row, the 178th row is given the 2-hydroxy-palmitic acid content increase of institute conversion plant.Therefore, in one embodiment, described protein or its homologue shown in Table II the 7th row, the 178th row are used to produce the 2-hydroxy-palmitic acid.

Be surprisingly found out that the yeast saccharomyces cerevisiae protein YFR042W transgene expression in Arabidopis thaliana shown in Table II the 3rd or 5 row, the 179th row is given the 2-hydroxyl tetracosene acid content increase of institute conversion plant.Therefore, in one embodiment, described protein or its homologue shown in Table II the 7th row, the 179th row are used to produce 2-hydroxyl tetracosenoic acid.

Be surprisingly found out that the yeast saccharomyces cerevisiae protein YOR317W transgene expression in Arabidopis thaliana shown in Table II the 3rd or 5 row, the 180th row is given hexadecadienoic acid (preferably Δ 7,10 hexadecadienoic acids) the content increase of institute conversions plant.Therefore, in one embodiment, described protein or its homologue shown in Table II the 7th row, the 180th row are used to produce hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids).

Be surprisingly found out that, give hiragonic acid (the preferred Δ 7 of institute conversion plant as Table II the 3rd or 5 row, the yeast saccharomyces cerevisiae protein YGL205W of 181-184 shown in capable and/or YIL150C and/or YKL132C and/or YOR344C and/or the e. coli k12 protein b3430 transgene expression in Arabidopis thaliana shown in Table II the 3rd or 5 row, the 185th row, 10,13 hiragonic acids) content increases.Therefore, in one embodiment, described protein or be used to produce hiragonic acid (preferred Δ 7,10,13 hiragonic acids) as its homologue shown in Table II the 7th row, 181-185 are capable.

Be surprisingly found out that the 2-hydroxy-palmitic acid content increase that the Escherichia coli protein b0161 shown in Table II A or IIB the 3rd or 5 row, the 558th, 559 row and/or b0758 transgene expression in Arabidopis thaliana are given institute conversion plant.Therefore, in one embodiment, described protein or its homologue shown in Table II the 7th row, the 558th, 559 row are used to produce the 2-hydroxy-palmitic acid.

Be surprisingly found out that as the Escherichia coli protein b0057 shown in Table II A or IIB the 3rd or 5 row, 560-563 are capable, b1097, b2078 or b3231 transgene expression in Arabidopis thaliana give the C24:1 fatty acid content increase of the plant that transforms.Therefore, in one embodiment, described protein or be used to produce C24:1 lipid acid as its homologue shown in Table II the 7th row, 560-563 are capable.

[0021.0.15.15] sees [0021.0.0.0]

The sequence of [0022.0.15.15] yeast saccharomyces cerevisiae YDR513W has been published in Jacq etc., Nature387 (6632 supplementary issue), 75-78 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be characterized as being be glutathione reductase, preferred glutaredoxin superfamily.Therefore, in one embodiment, the inventive method comprises the purposes with the active gene product of glutaredoxin superfamily (protein or its homologue that preferably have glutathione reductase activity) or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for producing fine chemicals in biological or its part as described, be margaric acid and/or the triglyceride level that comprises margaric acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase margaric acid and/or comprise the triglyceride level of margaric acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the margaric acid of preferably free or combining form.In another embodiment, YDR513W protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably participates in gene that lipid acid produces or coding lipid acid transporter protein with coding or is increased as the expression of gene of the compound in each lipid acid storehouse.In one embodiment, in the methods of the invention, the activity of glutaredoxin superfamily protein, preferred glutathione reductase is enhanced or produces, and for example the activity from the glutaredoxin superfamily protein of yeast saccharomyces cerevisiae, preferred glutathione reductase or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YER156C has been published in Dietrich, wait (Nature 387 (6632 supplementary issue), 78-81 (1997)), and its activity is not characterized still.As if it have the activity that is similar to Arabidopis thaliana putative protein matter F2K15.180.Therefore, in one embodiment, the inventive method comprises the purposes with yeast saccharomyces cerevisiae YER156C or the active protein of Arabidopis thaliana putative protein matter F2K15.180 or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be margaric acid and/or the triglyceride level that comprises margaric acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat and/or conjugate, preferably free or combining form.In another embodiment, YER156C protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably participates in gene that lipid acid produces or coding lipid acid transporter protein with coding or is increased as the expression of gene of the compound in each lipid acid storehouse.In one embodiment, in the methods of the invention, described activity for example YER156C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YER156C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YLR255C is by R; Johnson is committed to EMBL DataLibrary (February nineteen ninety-five), and its activity do not characterize yet, but preferably has the activity of yeast belong putative protein matter YLR255c superfamily.Therefore, in one embodiment, the inventive method comprises having the active gene product of yeast belong putative protein matter YLR255c superfamily, preferably have the gene product of YLR255C protein active or a purposes of its homologue from yeast saccharomyces cerevisiae as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, be margaric acid and/or the triglyceride level that comprises margaric acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase free or combining form quantity.In another embodiment, YLR255C protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably participates in gene that lipid acid produces or coding lipid acid transporter protein with coding or is increased as the expression of gene of the compound in each lipid acid storehouse.In one embodiment, in the methods of the invention, yeast belong putative protein matter YLR255c superfamily protein, preferred YLR255C activity of proteins are enhanced or produce, and for example the activity from the yeast belong putative protein matter YLR255c superfamily protein of yeast saccharomyces cerevisiae, preferred YLR255C protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b1829 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be heat shock protein with protease activity, preferred heat shock protein htpX superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having heat shock protein htpX superfamily (preferably from colibacillary, this protein has the activity of the heat shock protein with protease activity) purposes of active gene product or its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be margaric acid and/or the triglyceride level that comprises margaric acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase margaric acid and/or comprise the triglyceride level of margaric acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the margaric acid of preferably free or combining form.In another embodiment, b1829 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably participates in gene that lipid acid produces or coding lipid acid transporter protein with coding or is increased as the expression of gene of the compound in each lipid acid storehouse.In one embodiment, in the methods of the invention, the activity with heat shock protein of protease activity is enhanced or produces, and for example has the heat shock protein of protease activity or the activity of its homologue is enhanced or produces from colibacillary.

The sequence of yeast saccharomyces cerevisiae YER173W has been published in Dietrich etc., Nature387 (6632 supplementary issue), 78-81,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be to participate in dna damage and reduction division pachytene stage check position activatory check position protein.Therefore, in one embodiment, the inventive method comprises as shown here the have dna damage of participation and the gene product of reduction division pachytene stage check position activatory check position protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be 2-hydroxy-palmitic acid and/or the triglyceride level that comprises the 2-hydroxy-palmitic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat particularly increase the 2-hydroxy-palmitic acid of free or combining form and/or comprise the triglyceride level of 2-hydroxy-palmitic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, the quantity of oils and/or fat.In another embodiment, YER173w protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation glucosylceramide, a preferred glucocerebroside is increased.In one embodiment, in the methods of the invention, participate in dna damage and reduction division pachytene stage check position activatory check position activity of proteins and be enhanced or produce, for example be enhanced or produce from the participation dna damage of yeast saccharomyces cerevisiae and the activity of reduction division pachytene stage check position activatory check position protein or its homologue.

The sequence of yeast saccharomyces cerevisiae YFR042W has been published in Murakami etc., Nat.Genet.10 (3), 261-268 (1995) and Goffeau etc., Science 274 (5287), 546-547,1996, and have the supposition cell function of the possible membranin YFR042w superfamily of " cell survival institute proteins necessary ", preferably saccharomyces cerevisiae.Therefore, in one embodiment, the inventive method comprises as shown here having a possible active gene product of membranin YFR042w superfamily of yeast saccharomyces cerevisiae from yeast saccharomyces cerevisiae, preferably have the gene product of YFR042W protein active or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, it is tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) triglyceride level of (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat particularly increase tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise the triglyceride level of tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) of preferred free or combining form.In another embodiment, YFR042W protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation lipid, preferred cerebroside is increased.In one embodiment, in the methods of the invention, the YFR042W activity of proteins is enhanced or produces, and for example is enhanced or produces from the YFR042W protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YOR317W has been published in Dujon etc., Nature 387 (6632 supplementary issue), 98-102 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be characterized as being be the long-chain fat acyl: CoA synthetic enzyme, preferred longer chain fatty acid CoA ligase enzyme superfamily.Therefore, in one embodiment, the inventive method comprises that the active gene product of longer chain fatty acid CoA ligase enzyme superfamily that has from yeast saccharomyces cerevisiae as shown here (preferably has a long-chain fat acyl: the protein of CoA synthase activity or its homologue, for example as shown here) or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be hexadecadienoic acid (C16:2, preferred C16:2 Δ 7,10) and/or comprise the triglyceride level of hexadecadienoic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat particularly increase hexadecadienoic acid and/or comprise the triglyceride level of hexadecadienoic acid, lipid, oils and/or fat, the quantity of the hexadecadienoic acid of preferred free or combining form.In another embodiment, YOR317W protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation galactolipid, preferred single semi-lactosi diacylglycerol (DGDG) is increased.In one embodiment, in the methods of the invention, longer chain fatty acid CoA ligase enzyme superfamily protein, preferred long-chain fat acyl: the activity of CoA synthetic enzyme is enhanced or produces, for example from the longer chain fatty acid CoA ligase enzyme superfamily protein of yeast saccharomyces cerevisiae, preferred long-chain fat acyl: the activity of CoA synthetic enzyme or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YGL205W has been published in Tettelin etc.; Nature 387 (6632 supplementary issue); 81-84 (1997) and Goffeau etc.; Science 274 (5287); 546-547; 1996, and its cytoactive to be characterized as being be fatty acyl group coenzyme A oxydase, preferred ACOD superfamily.Therefore; in one embodiment; the inventive method comprises the purposes with the active gene product of ACOD superfamily (protein or its homologue that preferably has fatty acyl group coenzyme A oxidase activity as shown here) or its homologue from yeast saccharomyces cerevisiae as shown here; it is used for producing fine chemicals in biological or its part as described; be hiragonic acid (C16:3; preferred C16:2 Δ 7; 10; 13) and/or comprise the triglyceride level of hiragonic acid; lipid; preferably glycerine phosphatide; sphingolipid and/or galactolipid; oils and/or fat; particularly increase hiragonic acid and/or comprise the triglyceride level of hiragonic acid; lipid; preferably glycerine phosphatide; sphingolipid and/or galactolipid; oils and/or fat, the quantity of the hiragonic acid of preferably free or combining form.In another embodiment, YGL205W protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation galactolipid, preferred single semi-lactosi diacylglycerol (DGDG) is increased.In one embodiment; in the methods of the invention; ACOD superfamily protein, the preferred oxidasic activity of fatty acyl group coenzyme A are enhanced or produce, and for example the activity from the ACOD superfamily protein of yeast saccharomyces cerevisiae, preferred fatty acyl group coenzyme A oxydase or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Churcher etc., Nature387 (6632 supplementary issue), 84-87 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its as if having " the protein s phase (DNA synthetic) initial or finish institute must and/or chromobindins " the supposition cytoactive.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product with YIL150C protein active of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, it is hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or comprise hiragonic acid (preferred Δ 7,10,13 hiragonic acids) triglyceride level, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase hiragonic acid and/or comprise hiragonic acid, the triglyceride level of preferred hiragonic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the Δ 7 of preferred free or combining form, the quantity of 10,13 hiragonic acids.In another embodiment, YIL150C protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation galactolipid, preferred single semi-lactosi diacylglycerol (DGDG) is increased.In one embodiment, in the methods of the invention, the YIL150C activity of proteins is enhanced or produces, and for example is enhanced or produces from the YIL150C protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YKL132C has been published in Dujon etc., Nature 369 (6479), 371-378 (1994) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be characterized as being be the many glutamate synthases of leaf acyl, preferred many glutamate synthases of leaf acyl superfamily.Therefore, in one embodiment, the inventive method comprises the purposes with the active gene product of many glutamate synthases of leaf acyl superfamily (as shown here preferably have the active protein of the many glutamate synthases of leaf acyl or its homologue) or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for producing fine chemicals in biological or its part as described, it is hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or comprise the triglyceride level of hiragonic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or comprise the triglyceride level of hiragonic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the hiragonic acid of preferably free or combining form.In another embodiment, YKL132C protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation galactolipid, preferred single semi-lactosi diacylglycerol (DGDG) is increased.In one embodiment, in the methods of the invention, the activity of many glutamate synthases of leaf acyl superfamily protein, the preferred many glutamate synthases of leaf acyl is enhanced or produces, and for example the activity from many glutamate synthases of leaf acyl superfamily protein of yeast saccharomyces cerevisiae, the preferred many glutamate synthases of leaf acyl or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YOR344C has been published in Dujon etc., Nature 369 (6479), 371-378 (1994) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive has been characterized as being the protein that is rich in Serine of the involved in sugar glycolysis genetic expression that is supposition.Therefore, in one embodiment, the inventive method comprises the gene product of the protein active that is rich in Serine of the involved in sugar glycolysis genetic expression with supposition from yeast saccharomyces cerevisiae as shown here or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, it is hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or comprise hiragonic acid (preferred Δ 7,10,13 hiragonic acids) triglyceride level, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase hiragonic acid (the preferred Δ 7 of free or combining form, 10,13 hiragonic acids) and/or comprise hiragonic acid (preferred Δ 7,10,13 hiragonic acids) triglyceride level, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, the quantity of oils and/or fat.In another embodiment, YOR344C protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation galactolipid, preferred single semi-lactosi diacylglycerol (DGDG) is increased.In one embodiment, in the methods of the invention, the activity of proteins that is rich in Serine of the involved in sugar glycolysis genetic expression of supposing is enhanced or produces, and for example is enhanced or produces from the protein that is rich in Serine of the involved in sugar glycolysis genetic expression of the supposition of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b3430 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the Cori ester adenylyl transferase, preferred Cori ester adenylyl transferase superfamily.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of Cori ester adenylyl transferase superfamily (preferably from colibacillary, this protein has Cori ester adenylyl transferase activity) or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be hiragonic acid (C16:3, preferred C16:3 Δ 7,10,13) and/or comprise hiragonic acid, preferred Δ 7, the triglyceride level of 10,13 hiragonic acids, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat particularly increase hiragonic acid (the preferred Δ 7 of free or combining form, 10,13 hiragonic acids) and/or comprise the triglyceride level of hiragonic acid (preferred Δ 7,10,13 hiragonic acids), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, the quantity of oils and/or fat.In another embodiment, b3430 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation galactolipid, preferred single semi-lactosi diacylglycerol (DGDG) is increased.In one embodiment, in the methods of the invention, the activity of Cori ester adenylyl transferase is enhanced or produces, and for example the activity from colibacillary Cori ester adenylyl transferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b0057 has been published in Blattner, and Science 277 (5331), 1453-1474, and 1997, and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary b0057 protein or its homologue as shown here, as shown here, it is used for producing each fine chemicals in biological or its part, it is tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) triglyceride level of (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise the triglyceride level of tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) of preferably free or combining form.In another embodiment, b0057 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation lipid, preferred cerebroside is increased.In one embodiment, in the methods of the invention, the b0057 activity of proteins is enhanced or produces, and for example the activity from colibacillary or its homologue is enhanced or produces.

The sequence of e. coli k12 b0161 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be to have the active protein of pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b0161 of having protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be 2-hydroxy-palmitic acid and/or the triglyceride level that comprises the 2-hydroxy-palmitic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat particularly increase the 2-hydroxy-palmitic acid of free or combining form and/or comprise the triglyceride level of 2-hydroxy-palmitic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, the quantity of oils and/or fat.In another embodiment, b0161 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation glucosylceramide, a preferred glucocerebroside is increased.In one embodiment, in the methods of the invention, have the active activity of proteins of pericentral siphon serine protease (heat shock protein) and be enhanced or produce, for example have the active activity of proteins of pericentral siphon serine protease (heat shock protein) and be enhanced or produce from colibacillary.

The sequence of e. coli k12 b0758 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the protein with galactose-1-phosphate uridyl transferase active.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b0758 of having protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, be 2-hydroxy-palmitic acid and/or the triglyceride level that comprises the 2-hydroxy-palmitic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat particularly increase the 2-hydroxy-palmitic acid of free or combining form and/or comprise the triglyceride level of 2-hydroxy-palmitic acid, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, the quantity of oils and/or fat.In another embodiment, b0758 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation glucosylceramide, a preferred glucocerebroside is increased.In one embodiment, in the methods of the invention, activity of proteins with galactose-1-phosphate uridyl transferase active is enhanced or produces, and for example is enhanced or produces from colibacillary activity of proteins with galactose-1-phosphate uridyl transferase active.

The sequence of e. coli k12 b1097 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be to have the active protein of thymidylate kinase.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b1097 of having protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, it is tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) triglyceride level of (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise the triglyceride level of tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) of preferably free or combining form.In another embodiment, b1097 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation lipid, preferred cerebroside is increased.In one embodiment, in the methods of the invention, the b0057 activity of proteins is enhanced or produces, and for example the activity from colibacillary b0057 protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b2078 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be to have the active protein of histidine kinase.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b2078 of having protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, it is tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) triglyceride level of (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise the triglyceride level of tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) of preferably free or combining form.In another embodiment, b2078 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation lipid, preferred cerebroside is increased.In one embodiment, in the methods of the invention, the b2078 activity of proteins is enhanced or produces, and for example the activity from colibacillary b2078 protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b3231 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be to have the active protein of 50S ribosomal subunit protein matter L13.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b3231 of having protein active or the purposes of its homologue, as shown here, it is used for producing each fine chemicals in biological or its part, it is tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise tetracosenoic acid (Selacholeic acid, C24:1 Δ 15) triglyceride level of (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, particularly increase tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) and/or comprise the triglyceride level of tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid), lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or fat, the quantity of the tetracosenoic acid (Selacholeic acid) (preferred 2-hydroxyl tetracosenoic acid) of preferably free or combining form.In another embodiment, b3231 protein expression another expression of gene in the lipid biosynthetic pathway is increased, and preferably the proteinic expression of gene that produces with coding participation lipid, preferred cerebroside is increased.In one embodiment, in the methods of the invention, the b3231 activity of proteins is enhanced or produces, and for example the activity from colibacillary b3231 protein or its homologue is enhanced or produces.

The homologous compound (=homologue) of [0023.0.15.15] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, Table II the 3rd row, be respectively shown in the 174th, 175 or 176 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred margaric acid content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row, is respectively sequence shown in the 174th, 175 and/or 176 row.In one embodiment, Table II the 3rd row, be respectively shown in the 174th, 175 or 176 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 174th, 175 or 176 row from Ascomycota.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 174th, 175 or 176 row from yeast.In one embodiment, Table II the 3rd is listed as, is respectively the homologue of polypeptide shown in the 174th, 175 or 176 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 174th, 175 or 176 row are the homologues from Saccharomycetes.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 174th, 175 or 176 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the homologue that is respectively polypeptide shown in the 174th, 175 or 176 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred 2-hydroxy-palmitic acid content increases.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 178th, 558 or 559 row.In one embodiment, shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 178th, 558 or 559 row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II A or IIB the 3rd row, the 558th or 559 row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred 2-hydroxy-palmitic acid content increase.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 558th or 559 row.In one embodiment, shown in Table II A or IIB the 3rd row, the 558th or 559 row in the polypeptide homologue of any one from bacterium.In one embodiment, homologue is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 558th or 559 row is from γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 558th or 559 row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 558th or 559 row is from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 558th or 559 row is the homologue from Escherichia.

In one embodiment, shown in Table II A or IIB the 3rd row, the 179th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred 2-hydroxyl tetracosene acid content increases.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 179th row.In one embodiment, shown in Table II A or IIB the 3rd row, the 179th row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 179th row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 179th row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 179th row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 179th row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 179th row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 179th row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II A or IIB the 3rd row, the 180th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) content increases.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 180th row.In one embodiment, shown in Table II A or IIB the 3rd row, the 180th row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 180th row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 180th row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 180th row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 180th row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 180th row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 180th row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II A or IIB the 3rd row, be respectively shown in the 181st, 182,183 or 184 row that the homologue of any one is to have same or similar active homologue in the polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred hiragonic acid (preferred Δ 7,10,13 hiragonic acids) content increases.In one embodiment, homologue is the homologue that has Table I or II the 7th row, is respectively sequence shown in the 181st, 182,183 or 184 row.In one embodiment, Table II A or IIB the 3rd row, be respectively shown in the 181st, 182,183 or 184 row in the polypeptide homologue of any one from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II A or IIB the 3rd are listed as, are respectively the homologue of polypeptide shown in the 181st, 182,183 or 184 row from Ascomycota.In one embodiment, Table II A or IIB the 3rd are listed as, are respectively the homologue of polypeptide shown in the 181st, 182,183 or 184 row from yeast.In one embodiment, Table II A or IIB the 3rd are listed as, are respectively the homologue of polypeptide shown in the 181st, 182,183 or 184 row from the yeast guiding principle.In one embodiment, Table II A or IIB the 3rd row, the homologue that is respectively polypeptide shown in the 181st, 182,183 or 184 row are the homologues from Saccharomycetes.In one embodiment, Table II A or IIB the 3rd row, the homologue that is respectively polypeptide shown in the 181st, 182,183 or 184 row are to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II A or IIB the 3rd row, the homologue that is respectively polypeptide shown in the 181st, 182,183 or 184 row are to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, shown in Table II A or IIB the 3rd row, the 177th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred margaric acid content increase.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 177th row.In one embodiment, shown in Table II A or IIB the 3rd row, the 177th row in the polypeptide homologue of any one from bacterium.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 177th row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 177th row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 177th row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 177th row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 177th row is to have same or similar activity and from the homologue of Escherichia.

In one embodiment, shown in Table II A or IIB the 3rd row, the 185th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, the increase of preferred hiragonic acid (preferred Δ 7,10,13 hiragonic acids) content.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 185th row.In one embodiment, shown in Table II A or IIB the 3rd row, the 185th row in the polypeptide homologue of any one from bacterium.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 185th row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 185th row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 185th row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 185th row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 185th row is to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II A or IIB the 3rd row, 560-563 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, the active raising given each fine chemicals in the biology, preferred C24:1 lipid acid, preferred C24:1 Δ 15 fatty acid content increase.In one embodiment, homologue be have Table I or II the 7th row, 560-563 capable shown in the homologue of sequence.In one embodiment, Table II A or IIB the 3rd row, 560-563 capable shown in the polypeptide any one homologue from bacterium.In one embodiment, Table II A or IIB the 3rd row, 560-563 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II A or IIB the 3rd row, 560-563 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II A or IIB the 3rd row, 560-563 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II A or IIB the 3rd row, 560-563 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II A or IIB the 3rd row, 560-563 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

[0023.1.15.15] as the homologue of the polypeptide shown in Table II A or IIB the 3rd row, 174-185,558-563 are capable can be by Table I A or IB the 7th row, be respectively 174-185,558-563 capable shown in nucleic acid molecule encoding polypeptide or can be Table II A or IIB the 7th row, be respectively 174-185,558-563 capable shown in polypeptide.As the homologue of polypeptide shown in Table II A or IIB the 3rd row, 174-185,558-563 are capable can be by Table I A or IB the 7th row, 174-185,558-563 capable shown in nucleic acid molecule encoding polypeptide or can be Table II A or IIB the 7th row, 174-185,558-563 capable shown in polypeptide.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, 174-177 are capable can be have increase margaric acid content and/or quantity is active, by Table I A or IB the 7th row, 174-177 capable shown in the polypeptide of nucleic acid molecule encoding or Table II A or IIB the 7th row, 174-177 capable shown in polypeptide.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 179th row can be have increase 2-hydroxyl tetracosene acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 179th row or Table II A or IIB the 7th row, the 179th row.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 180th row can be have increase hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 180th row or Table II A or IIB the 7th row, the 180th row.

The homologue of polypeptide can be to have increase hiragonic acid (preferred Δ 7 shown in Table II A or IIB the 3rd row, 181-185 were capable, 10,13 hiragonic acids) content and/or quantity active, by Table I A or IB the 7th row, 181-185 capable shown in the polypeptide of nucleic acid molecule encoding or Table II A or IIB the 7th row, 181-185 capable shown in polypeptide.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 558th, 559 row can be have increase 2-hydroxy-palmitic acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 558th, 559 row or Table II A or IIB the 7th row, the 558th, 559 row.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, 560-563 are capable can be have increase C24:1 lipid acid, preferred C24:1 Δ 15 fatty acid content and/or quantity is active, by Table I A or IB the 7th row, 560-563 capable shown in the polypeptide of nucleic acid molecule encoding or Table II A or IIB the 7th row, 560-563 capable shown in polypeptide.

[0024.0.0.15] sees [0024.0.0.0]

[0025.0.15.15] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause biology or its part, the lipid acid level increases in the preferred described biomass cells, particularly margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid and/or 2-hydroxy-palmitic acid and/or C24:1 lipid acid, preferred Δ 7,10 hexadecadienoic acids and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) level increases, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example to show HA or IIB the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, activity of proteins shown in 559 row and/or 560-563 are capable.In preferred embodiments, protein or polypeptide have Table II A or IIB the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in proteinic above-mentioned extra activity.In whole specification sheets, if this kind protein or polypeptide still have Table II the 3rd row, be respectively Table II A or IIB the 3rd row, 174-177 capable and/or the 178th the row and/or the 179th the row and/or the 180th the row and/or 181-185 capable shown in any one proteinic biology or the enzyme activity, if promptly with Table II A or IIB the 3rd row, 174-176 or 178 179 or 180 or 181-184 capable shown in any one yeast saccharomyces cerevisiae protein compare and/or with Table II A or IIB the 3rd row, the 177th or 185 or 558,559 or 560-563 capable shown in any one e. coli k12 protein compare, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I A or IB the 4th row and is expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as increase each fine chemicals in biological or its part.For example play eozoan and express biologically, and play eozoan with biological shown in Table I A or IB the 4th row from identical section, order, guiding principle or door from different sections, order, guiding principle or door.

[0026.0.0.15] to [0033.0.0.15] sees that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.15.15] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, it has Table II A or IIB the 3rd row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, protein shown in 559 row and/or 560-563 are capable, or by Table I A or IB the 5th row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the protein of nucleic acid molecule encoding shown in 559 row and/or 560-563 are capable, or its homologue is (as Table I the 7th row, 174-177 capable and/or the 178th row and/or the 179th row and/or 181-185 is capable and/or the 558th, homologue shown in 559 row and/or 560-563 are capable) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore it shows the fine chemicals quantity that increases.

[0035.0.0.15] to [0036.0.0.15] sees that [0035.0.0.0] is to [0036.0.0.0]

There is a series of mechanism in [0037.0.15.15], can directly or indirectly influence output, production and/or the production efficiency of lipid acid by the modification of these machine-made egg white matters (polypeptide for example of the present invention).

[0038.0.0.15] to [0044.0.0.15] sees that [0038.0.0.0] is to [0044.0.0.0]

[0045.0.15.15] in one embodiment, at yeast saccharomyces cerevisiae protein YDR513W or its homologue, for example under the situation that the activity of glutathione reductase, preferred glutaredoxin superfamily (shown in Table II the 5th or 7 row, the 174th row) is enhanced, give each fine chemicals, preferred margaric acid be increased in 24% and 97% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER156C or its homologue, for example under the activity situation about being enhanced of YER156C protein (shown in Table II the 5th or 7 row, the 175th row), give each fine chemicals, preferred margaric acid be increased in 20% and 49% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173W or its homologue, for example participate under the activity situation about being enhanced of dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table II the 5th or 7 row, the 178th row), give each fine chemicals, preferred 2-hydroxy-palmitic acid be increased in 26% and 394% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFR042W or its homologue, for example under the situation that the activity of the membranin YFR042w superfamily that cell survival institute proteins necessary, preferably saccharomyces cerevisiae are possible (shown in Table II the 5th or 7 row, the 179th row) is enhanced, give each fine chemicals, preferred 2-hydroxyl tetracosenoic acid be increased in 28% and 56% or more between.

In one embodiment; at yeast saccharomyces cerevisiae protein YGL205W or its homologue; for example under the situation that the activity of fatty acyl group coenzyme A oxydase, preferred ACOD superfamily (shown in Table II the 5th or 7 row, the 181st row) is enhanced; give each fine chemicals, preferred hiragonic acid (preferred Δ 7; 10,13 hiragonic acids) be increased in 14% and 16% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) is initial or finish under the situation that the activity of institute's proteins necessary and/or chromobindins (shown in Table II the 5th or 7 row, the 182nd row) is enhanced, give each fine chemicals, preferred hiragonic acid (preferred Δ 7,10,13 hiragonic acids) be increased in 150% and 205%, preferred 224% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YKL132C or its homologue, for example under the situation that the activity of the many glutamate synthases of leaf acyl, preferred many glutamate synthases of leaf acyl superfamily (shown in Table II the 5th or 7 row, the 183rd row) is enhanced, give each fine chemicals, preferred hiragonic acid (preferred Δ 7,10,13 hiragonic acids) be increased in 13% and 56% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YLR255C or its homologue, for example under the activity situation about being enhanced of yeast belong putative protein matter YLR255c superfamily (shown in Table II the 5th or 7 row, the 176th row), give each fine chemicals, preferred margaric acid be increased in 20% and 27% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR317W or its homologue, long-chain fat acyl for example: under the situation that the activity of CoA synthetic enzyme, preferred longer chain fatty acid CoA ligase enzyme superfamily (shown in Table II the 5th or 7 row, the 180th row) is enhanced, give each fine chemicals, preferred hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) be increased in 19% and 69% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR344C or its homologue, for example under the activity situation about being enhanced of the protein that is rich in Serine of Jia Ding involved in sugar glycolysis genetic expression (shown in Table II the 5th or 7 row, the 184th row), give each fine chemicals, preferred hiragonic acid (preferred Δ 7,10,13 hiragonic acids) be increased in 12% and 20% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, for example have under the situation that the activity of the heat shock protein of protease activity, preferred heat shock protein htpX superfamily (shown in Table II the 5th or 7 row, the 177th row) is enhanced, give each fine chemicals, preferred margaric acid be increased in 20% and 133% or more between.

In one embodiment, at e. coli k12 protein b3430 or its homologue, for example under the situation that the activity of the transcriptional of Cori ester adenylyl transferase, preferred Cori ester adenylyl transferase superfamily (shown in Table II the 5th or 7 row, the 185th row) is enhanced, give each fine chemicals, preferred hiragonic acid (preferred Δ 7,10,13 hiragonic acids) be increased in 12% and 20% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b0057 or its homologue (shown in Table II the 5th or 7 row, the 560th row) is enhanced, give each fine chemicals, preferred C24:1 lipid acid be increased in 22% and 37% or more between.

In one embodiment, at e. coli k12 protein b0161 or its homologue, for example under the activity situation about being enhanced of pericentral siphon serine protease (heat shock protein) (shown in Table II the 5th or 7 row, the 558th row), give each fine chemicals, preferred 2-hydroxy-palmitic acid be increased in 21% and 48% or more between.

In one embodiment, at e. coli k12 protein b0758 or its homologue, for example under the activity situation about being enhanced of galactose-1-phosphate uridyl transferase protein (shown in Table II the 5th or 7 row, the 559th row), give each fine chemicals, preferred 2-hydroxy-palmitic acid be increased in 19% and 38% or more between.

In one embodiment, at e. coli k12 protein b1097 or its homologue, for example under the activity situation about being enhanced of thymidylate kinase protein (shown in Table II the 5th or 7 row, the 561st row), give each fine chemicals, preferred C24:1 lipid acid be increased in 23% and 41% or more between.

In one embodiment, at e. coli k12 protein b2078 or its homologue, for example under the activity situation about being enhanced of the sensibility histidine kinase protein in the two-pack regulator control system (shown in Table II the 5th or 7 row, the 562nd row), give each fine chemicals, preferred C24:1 lipid acid be increased in 23% and 41% or more between.

In one embodiment, at e. coli k12 protein b3231 or its homologue, for example under the activity situation about being enhanced of 50S ribosomal subunit protein matter L13 protein (shown in Table II the 5th or 7 row, the 563rd row), give each fine chemicals, preferred C24:1 lipid acid be increased in 23% and 48% or more between.

[0046.0.15.15] in one embodiment, yeast saccharomyces cerevisiae protein YDR513W or its homologue, for example the activity of glutathione reductase, preferred glutaredoxin superfamily (shown in Table II the 5th or 7 row, the 174th row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YER156C or its homologue, for example the activity of YER156C protein (shown in Table II the 5th or 7 row, the 175th row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YER173W or its homologue, the activity that for example participates in dna damage and reduction division pachytene stage check position activatory check position protein (shown in Table II the 5th or 7 row, the 178th row) are given each fine chemicals and Geng Duo and are had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YFR042W or its homologue, for example the activity of the membranin YFR042w superfamily that cell survival institute proteins necessary, preferably saccharomyces cerevisiae are possible (shown in Table II the 5th or 7 row, the 179th row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment; yeast saccharomyces cerevisiae protein YGL205W or its homologue, for example the activity of fatty acyl group coenzyme A oxydase, preferred ACOD superfamily (shown in Table II the 5th or 7 row, the 181st row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example S phase (DNA synthetic) activity initial or that finish institute's proteins necessary and/or chromobindins (shown in Table II the 5th or 7 row, the 182nd row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YKL132C or its homologue, for example the activity of the many glutamate synthases of leaf acyl, preferred many glutamate synthases of leaf acyl superfamily (shown in Table II the 5th or 7 row, the 183rd row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YLR255C or its homologue, for example the activity of yeast belong putative protein matter YLR255c superfamily (shown in Table II the 5th or 7 row, the 176th row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YOR317W or its homologue, for example long-chain fat acyl: the activity of CoA synthetic enzyme, preferred longer chain fatty acid CoA ligase enzyme superfamily (shown in Table II the 5th or 7 row, the 180th row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, yeast saccharomyces cerevisiae protein YOR344C or its homologue, for example the activity of the protein that is rich in Serine of Jia Ding involved in sugar glycolysis genetic expression (shown in Table II the 5th or 7 row, the 184th row) is given each fine chemicals and Geng Duo and is had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, e. coli k12 protein b1829 or its homologue, the activity that for example has a heat shock protein of protease activity, preferred heat shock protein htpX superfamily (shown in Table II the 5th or 7 row, the 177th row) are given each fine chemicals and Geng Duo and are had the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid or its precursor and increase.

In one embodiment, e. coli k12 protein b3430 or its homologue, for example the activity of Cori ester adenylyl transferase, preferred Cori ester adenylyl transferase superfamily transcriptional (shown in Table II the 5th or 7 row, the 185th row) is given each fine chemicals and Geng Duo has the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, compound or its precursor increase.

In one embodiment, the activity of e. coli k12 protein b0057 or its homologue (shown in Table II the 5th or 7 row, the 560th row) gives that each fine chemicals and Geng Duo have the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, compound or its precursor increase.

In one embodiment, e. coli k12 protein b0161 or its homologue, for example the activity of pericentral siphon serine protease (heat shock protein) active (shown in Table II the 5th or 7 row, the 558th row) is given each fine chemicals and Geng Duo has the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, compound or its precursor increase.

In one embodiment, e. coli k12 protein b0758 or its homologue, for example the activity of galactose-1-phosphate uridyl transferase active (shown in Table II the 5th or 7 row, the 559th row) gives that each fine chemicals and Geng Duo have the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, compound or its precursor increase.

In one embodiment, e. coli k12 protein b1097 or its homologue, for example the activity of thymidylate kinase activity (shown in Table II the 5th or 7 row, the 561st row) gives that each fine chemicals and Geng Duo have the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, compound or its precursor increase.

In one embodiment, e. coli k12 protein b2078 or its homologue, for example the activity of the sensibility histidine kinase activity in the two-pack regulator control system (shown in Table II the 5th or 7 row, the 562nd row) gives that each fine chemicals and Geng Duo have the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, compound or its precursor increase.

In one embodiment, e. coli k12 protein b3231 or its homologue, for example the activity of 50S ribosomal subunit protein matter L13 activity (shown in Table II the 5th or 7 row, the 563rd row) gives that each fine chemicals and Geng Duo have the active compound of lipid acid or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, compound or its precursor increase.

[0047.0.0.15] and [0048.0.0.15] sees [0047.0.0.0] and [0048.0.0.0]

[0049.0.15.15] has to give and improves margaric acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:15686 as SEQ ID, 15687 and/or 15688 or 19697,15698,15699,15700,15701 and/or 15702 or 15703, the sequence of consensus sequence shown in 15704 and/or 15705 and/or as Table IV the 5th or 7 row, the sequence of consensus sequence shown in 174-177 is capable and/or as Table II the 5th or 7 row, the sequence of polypeptide shown in 174-177 is capable or its function homologue described in the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 174-177 is capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the margaric acid level of giving described in the literary composition increases.

Have to give and improve 2-hydroxyl tetracosenoic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the sequence of consensus sequence shown in SEQ ID NO:15691 and/or 15692 and/or are listed as Table IV the 5th or 7, the sequence of consensus sequence shown in the 179th row and/or as Table II the 5th or 7 be listed as, the sequence of polypeptide or its function homologue described in the literary composition shown in the 179th row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 179th row) sequence of coded polypeptide, and have the activity that the 2-hydroxyl tetracosenoic acid level of giving described in the literary composition increases.

Have to give and improve hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:15661 as SEQ ID, 16662,15663,15664, the sequence of consensus sequence shown in 15665 and/or 15666 and/or as Table IV the 5th or 7 row, the sequence of consensus sequence shown in the 180th row and/or as Table II the 5th or 7 be listed as, the sequence of polypeptide or its function homologue described in the literary composition shown in the 180th row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 180th row) sequence of coded polypeptide, and has the activity of giving the increase of hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) level described in the literary composition.

Have to give and improve hiragonic acid (preferred Δ 7,10,13 hiragonic acids) quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as SEQ ID NO:15706 and/or 15707 or 15689 and/or, 15690 or 15682,15683,15684 and/or, 15685 or 15658,15659 and/or 15660 or 15677,15678,15679, the sequence of consensus sequence shown in 15680 and/or 15681 and/or as Table IV the 5th or 7 row, the sequence of consensus sequence shown in 181-185 is capable and/or as Table II the 5th or 7 row, the sequence of polypeptide shown in 181-185 is capable or its function homologue described in the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 181-185 is capable or its function homologue as herein described) sequence of coded polypeptide, and have and give hiragonic acid (preferred Δ 7 described in the literary composition, 10,13 hiragonic acids) activity of level increase.

Have to give and improve 2-hydroxy-palmitic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:85367 as SEQ ID, 85368,85369,85370,85371,85372 or 85515,85516,85517, the sequence of consensus sequence shown in 85518 and/or as Table IV the 5th or 7 row, the sequence of consensus sequence shown in the 558th or 559 row and/or as Table II the 5th or 7 be listed as, the sequence of polypeptide or its function homologue described in the literary composition shown in the 558th or 559 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 558th or 559 row) sequence of coded polypeptide, and have the activity that the 2-hydroxy-palmitic acid level of giving described in the literary composition increases.

Have to give and improve C24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:85801 as SEQ ID, 85802,85803 or 86104,86105,86106 or 86427,86428, the sequence of consensus sequence shown in 86429 and/or as Table IV the 5th or 7 row, the sequence of consensus sequence shown in 560-563 is capable and/or as Table II the 5th or 7 row, the sequence of polypeptide shown in 560-563 is capable or its function homologue described in the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 560-563 is capable or its function homologue as herein described) sequence of coded polypeptide, and have and give C24:1 lipid acid (the preferred C24:1 Δ 15 lipid acid) activity that level increases described in the literary composition.

[0050.0.15.15] for the purposes of the present invention, term " margaric acid ", " 2-hydroxy-palmitic acid ", " 2-hydroxyl tetracosenoic acid ", " hexadecadienoic acid " (preferred " Δ 7; 10 hexadecadienoic acids ") and/or " hiragonic acid " (preferred " Δ 7; 10; 13 hiragonic acids ") and/or C24:1 lipid acid also comprise corresponding salt, for example, the salt of the sylvite of above-mentioned lipid acid or sodium salt or the above-mentioned lipid acid that has amine (as diethylamine) or the ester of above-mentioned lipid acid.

[0051.0.15.15] because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains fine chemicals (i.e. raising amount free or in conjunction with chemical), for example fatty acid composition.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of multiple lipid acid.

[0052.0.0.15] sees [0052.0.0.0]

[0053.0.15.15] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention (for example has as Table II the 3rd row, protein or its homologue shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable (for example Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) active polypeptide) express increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention (for example have as Table II the 3rd row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 103-106 and/or 468-471 is capable or the 107th and/or 108 row shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or the of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, protein or its homologue shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable (for example Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by the coded protein of nucleic acid molecule of the present invention, polypeptide perhaps of the present invention (for example has as Table II the 3rd row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, (for example Table II the 5th or 7 is listed as for protein or its homologue shown in 559 row and/or 560-563 are capable, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) active polypeptide) express and improve;

(e) by add the activity that one or more external source inducible factors come stimulating protein in biological or its part, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, protein or its homologue shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable (for example Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) active polypeptide) express and improve;

(f) transgenosis of expression coded protein, described protein is given the activity with each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, protein or its homologue shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable (for example Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, and described nucleic acid molecule encoding has the active of each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, protein or its homologue shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable (for example Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) active polypeptide);

(h) by adding positive Expression element or removing negative Expression element and improve code book invention polypeptide and (for example have as Table II the 3rd row, protein or its homologue shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable (for example Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) active polypeptide) the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) (heat shock protein(HSP) for example of the present invention) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.15.15] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having according to Table II the 3rd row, protein or its homologue shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable (for example Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide shown in 559 row and/or 560-563 are capable) giving each fine chemicals after active expression of polypeptides or the activity increases.

[0055.0.0.15] to [0067.0.0.15] sees that [0055.0.0.0] is to [0067.0.0.0]

[0068.0.15.15] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of lipid acid.

The adjusting of [0069.0.15.15] gene or its gene product is subjected to less influence and is interpreted as enzymic activity is regulated decline, causes the specific activity of gene or its product or the raising of cytoactive.The activity that the raising of enzymic activity can be regarded as enzyme improves at least 10% with initial biophase ratio, advantageously is at least 20%, 30% or 40%, particularly advantageously is at least 50%, 60% or 70%.This causes that desired fats acid productivity improves.

[0070.0.15.15] imports biology separately or with other assortment of genes owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example contain high level (from the physiology of nutrition angle) lipid acid for example margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) favourable fatty acid composition.

[0071.0.15.15] preferably, with composition of the present invention or composition biological or that its part provides also contain higher amount to the nutrition of animal and human's class or healthy have the metabolite that just influencing or low quantity to nutrition of animal and human's class or healthy metabolite with negatively influencing.Similarly, can improve the number or the activity that input or output other required genes of nutrition or metabolite (comprising the required amino acid of the synthetic fine chemicals of cell biological, lipid acid, VITAMIN etc. and precursor thereof), to improve the concentration of necessity in cell or the corresponding storage compartment or associated precursors, cofactor or intermediate product.Because the raising or the active new generation of polypeptide active of the present invention, perhaps, can in host living beings such as plant or microorganism, improve output, production and/or the generation efficient of fine chemicals owing to nucleic acid sequence of the present invention and/or to participating in the raising of biosynthetic other generegulation of fine chemicals (activity of for example passing through to improve the activity of the enzyme that synthesizes precursor or participating in one or more genes of fine chemicals decomposition by destruction).

[0072.0.15.15] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds is except margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids); Comprise margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) outside triacylglycerol, lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid, oils and/or the fat, also has for example other lipid acid of some compounds, as palmitinic acid, stearic acid, Zoomeric acid, oleic acid, linolic acid and/or linolic acid or sinapinic acid and/or arachidonic acid.

[0073.0.15.15] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or or its homologue (for example Table II the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of promptly giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or bonded fine chemicals by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and other optional free and/or bonded lipid acid.

[0074.0.15.15] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or bonded fine chemicals or free and bonded fine chemicals, and if dissociate or/and bonded lipid acid as selecting to produce, reclaim and expect to separate other.

[0075.0.0.15] to [0077.0.0.15] sees that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.15.15] can directly be processed into biological (isolating in case of necessity) fine chemicals as microorganism or plant or recovery food or animal-feed or be used for other application subsequently, for example carries out according to EP-A-0 568 608, EP-A-568 606, WO 2004/007732, WO 02/057465, WO01/02591, WO 2004/071467 or the disclosed content of US 20020156254 (this paper is incorporated herein by reference especially).Can pass through highly basic hydrolysis, extraction and crystallization or by thin-layer chromatography with those skilled in the art are known and hereinafter described additive method, in a usual manner purified fermentation broth, tunning, plant or plant product.These are different checks that the product of steps is lipid acid or the fatty acid composition that still contains fermented liquid, vegetable particle and different quantities cellular component, and the favourable fermented liquid and the scope of cellular component are 0 to 99% by weight, preferably are lower than 80%, especially preferably are lower than 50%.

[0079.0.0.15] to [0084.0.0.15]: see that [0079.0.0.0] is to [0084.0.0.0]

[0085.0.15.15] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, 174-177 is capable and/or the 178th the row and/or the 179th the row and/or the 180th the row and/or 181-185 is capable and/or the 558th, 559 the row and/or 560-563 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.15] to [0087.0.0.15] sees that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.15.15] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified fatty acid content.Because the nutritive value that for example is used for the plant of feeding animals depends on above-mentioned lipid acid and as the total number of the lipid acid in fodder energy source, so this is very important for the plant breeder.After above-mentioned protein active has been enhanced or has produced, perhaps after nucleic acid molecule according to the present invention or polypeptide expression have been produced or have been improved, the transgenic plant that produced grow on the nutritional medium or among or grow in the soil results subsequently.

[0089.0.0.15] to [0095.0.0.15] sees that [0089.0.0.0] is to [0095.0.0.0]

[0096.0.15.15] is in another embodiment preferred of the present invention, improve nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide, for example lipid acid transporter protein or compound (as the pond of desired fats acid in the biology) are combined and be can be used for producing fine chemicals and (see Bao and Ohlrogge, Plant Physiol.1999 August; 120 (4): 1057-1062).The proteinic effect of this lipid acid transporter is to connect synthetic place of lipid acid and so-called stored tissue (depot fat acid therein, triacylglycerol, oils and fat).

[0097.0.0.15] sees that the content of [0097.0.0.0] increase bonded fine chemicals also is favourable.

[0098.0.15.15] in preferred embodiments, fine chemicals (margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids)) be produce according to the present invention and carry out isolating where necessary.The mixture that the method according to this invention produces other lipid acid such as Zoomeric acid, palmitinic acid, stearic acid, oleic acid, linolic acid, Selacholeic acid and/or linolenic acid mixture or other lipid acid is favourable.

For microbial fermentation, aforementioned fatty acids can be accumulated in substratum and/or the cell [0099.0.15.15].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps separation of fatty acids or fatty acid composition from biology and substratum.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.15.15] comprises the transgenic plant that the method according to this invention synthetic comprises lipid acid such as saturated or polyunsaturated fatty acid and can advantageously directly put on market, and do not need institute's synthetic oils, lipid or lipid acid are separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet, the fine chemicals that produces according to the inventive method can also from biological, advantageously from plant with the isolated in form of oils, fat, lipid and/or free fatty acids.Yet the fine chemicals that the method according to this invention produces can also be with the form of the form of its oils, fat, lipid and/or free fatty acids from biology, advantageously separate from plant.Can obtain the lipid acid that produces by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part, preferred plant seed.In order to improve the efficient that oil extracts, with vegetable material, particularly seed clean, softening (temper) and to shell in case of necessity and peel off be favourable.In this article, oils, fat, lipid and/or free fatty acids can need not heat cold be beaten or cold-press process obtains by being called.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.In this mode, can separate more than 96% of compound that the inventive method produces.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.Afterwards, randomly, free fatty acids can be by advantageously using down alkali (as water-soluble KOH or NaOH) and acid hydrolysis processing or remove by enzymic hydrolysis at alcohol (for example methyl alcohol and ethanol), and by for example being separated and subsequently acidifying (is for example passed through H ₂SO ₄) separate.Can also not use above-mentioned procedure of processing and release fat acid.If expectation can water thoroughly be washed products therefrom to remove residual soap and alkali in the product, and is dry then.Be to remove residual pigment in the product, can use Fuller's earth (filler ' s earth) or activated carbon bleaching.At last under vacuum vapor distillation with the product deodorizing.The lipid acid of these chemical purifications or fatty acid composition can advantageously be applied to grocery trade, cosmetic industry, particularly pharmacy industry.

[0101.0.15.15] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.15.15] for example, lipid acid can advantageously detect by the GC separation method.By using standard method of analysis GC, GC-MS or TLC can clearly detect the existence of fatty acids products to the biology analysis of recombinating, described analytical procedure Christie and reference wherein are described (1997, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric method], Lipide33:343-353).An embodiment is by FAME and GC-MS or TLC (abbreviation: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography) analysis lipid acid.By ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material fragmentation to be analyzed.After the fragmentation, must material is centrifugal.With pellet resuspended in distilled water, in 100 ℃ of heating 10 minutes, cooled on ice was also centrifugal, then extracts 1 hour in 90 ℃ in containing the 0.5M sulfuric acid (in methyl alcohol) of 2% Propanal dimethyl acetal, this causes producing the oil and the fat compound of hydrolysis, and this can obtain transmethylase fat.In sherwood oil, extract these fatty acid methyl esters and finally use capillary column (Chrompack, the WCOT fused silica, CP-Wax-52 CB, 25 μ m, 0.32mm) gradient temperature between 170 ℃ and 240 ℃ 20 minutes and 240 ℃ carried out the GC analysis in following 5 minutes.The identity of resulting fatty acid methyl ester must be used and can define from the standard that commercial source (being Sigma) obtains.

[0102.1.15.15] another analytical procedure is by descriptions (Proceedings of the OceanDrilling Program, Scientific Results Volume 169,2000) such as Summit.Use has the capillary gas chromatography fatty acid methyl ester that flame ionization detects.By half selectivity HSQC (the single quantum coherent of heteronuclear)-NMR (Willker etc., Magn.Reson.Chem.36, S79-S84 (1998)) can further distinguish and quantitative multiple monounsaturated fatty acids and the polyunsaturated fatty acid that does not carry out in the previous isolating sample.

Use be connected with high performance liquid chromatography (HPLC) combination in-source (taper hole voltage) fragmentation, the mass spectral HPLC/ESI-MS of negatively charged ion electron spray ionisation (ESI) can study fatty acid composition, preferred single semi-lactosi diacylglycerol (DGDG) (MGDG) and digalactosyl diacylglycerol (DGDG) ester (DGDG) (Kim etc., Bull.Korean Chem.Soc.2003, Vol.24, No.8).

[0103.0.15.15] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferred encoding mature form at least as Table II the 5th or 7 row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in polypeptide or its segmental nucleic acid molecule; Described nucleic acid molecule is given fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule as Table I A or IB the 5th or 7 row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use as Table III the 7th row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise as Table IV the 7th row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in consensus sequence and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0104.0.15.15] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A or IB the 5th or 7 row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the sequence difference be one or more Nucleotide, perhaps not by Table I A or IB the 5th or 7 row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 174-177 is capable and/or the 178th the row and/or the 179th the row and/or the 180th the row and/or 181-185 is capable and/or the 558th, 559 the row and/or 560-563 capable shown in polypeptide of sequence.

[0105.0.0.15] to [0107.0.0.15] sees that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.15.15] method of the present invention and had Table I A or IB the 5th or 7 row, the nucleic acid molecule of sequence shown in capable and/or the 178th row of 174-177 and/or the 179th row and/or the 180th row and/or 181-185 are capable, from Table II the 5th or 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, aminoacid sequence shown in 559 row and/or 560-563 are capable is derived or is listed as from containing Table IV the 5th or 7,174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 559 row and/or 560-563 are capable, perhaps its coding has as Table I A or IB the 3rd row, 5 or 7, the 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the enzymic activity of polypeptide or bioactive polypeptide shown in 559 row and/or 560-563 are capable or for example give derivative or the homologue that each fine chemicals increases behind its expression or active the increasing.

[0109.0.15.15] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make the lipid acid that the inventive method produced be optimized synthetic.

[0110.0.0.15] sees [0110.0.0.0]

[0111.0.0.15] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.15.15] the inventive method is the isolated nucleic acid sequences form, its coding has as Table I A or IB the 3rd row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 the row and/or 560-563 capable shown in polypeptide active polypeptide or have as Table II the 5th and 7 row, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the polypeptide of peptide sequence shown in 559 row and/or 560-563 are capable, and give each fine chemicals level increase.

[0113.0.0.15] to [0120.0.0.15] sees that [0113.0.0.0] is to [0120.0.0.0]

[0121.0.15.15] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, peptide sequence or its functional homologue as herein described had the difference of one or more amino acid moleculars shown in 559 row and/or 560-563 were capable, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, giving the margaric acid level shown in 174-177 is capable after the activity of peptide sequence increases, perhaps improving Table II the 5th or 7 row, the 178th or 558, giving 2-hydroxy-palmitic acid level after the activity of peptide sequence shown in 559 row increases, perhaps improving Table II the 5th or 7 row, giving 2-hydroxyl tetracosenoic acid level after the activity of peptide sequence shown in the 179th row increases, perhaps improving Table II the 5th or 7 row, give hexadecadienoic acid (preferred Δ 7 shown in the 180th row after the activity of peptide sequence, 10 hexadecadienoic acids) level increases, perhaps improving Table II the 5th or 7 row, give hiragonic acid (preferred Δ 7 shown in 181-185 is capable after the activity of peptide sequence, 10,13 hiragonic acids) level increases, and is perhaps improving Table II the 5th or 7 row, giving C24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) level shown in 560-563 is capable after the activity of peptide sequence increases.

[0122.0.0.15] to [0127.0.0.15] sees that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.15.15] polymerase chain reaction (PCR) amplification is used is (for example as Table III the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the primer of 560-563 shown in capable to) can be based on sequence shown in this paper, for example Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable or from as Table II the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence deutero-sequence produced shown in 559 row and/or 560-563 were capable.

[0129.0.15.15] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, be respectively 174-177 capable and/or and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in consensus sequence from described comparison.

[0130.0.15.15] can use degenerated primer to have aforementioned activity by pcr amplification then and (give lipid acid as comprise this segmental protein expression or activity in raising after, margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 for example, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) increase) the new protein fragment.

[0131.0.0.15] to [0138.0.0.15] sees that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.15.15] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna is listed as with Table I A or IB the 5th or 7 under loose hybridization conditions, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence hybridization shown in 559 row and/or 560-563 are capable, and the coding expression has the margaric acid of increasing and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the active peptide of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid).

[0140.0.0.15] to [0146.0.0.15]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.15.15] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred table B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, one of nucleotide sequence complementary nucleic acid molecule was meant these molecules shown in 559 row and/or 560-563 were capable: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.15.15] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, nucleotide sequence or its portion homologous were at least about 30% shown in 559 row and/or 560-563 were capable, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably has an aforementioned activity, particularly have and after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increase margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the activity of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid).

[0149.0.15.15] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, one of nucleotide sequence shown in 559 row and/or 560-563 are capable or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and (as gives each fine chemicals, margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 for example, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) increase) and randomly as Table II A or IIB the 5th be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the protein of protein active shown in 559 row and/or 560-563 are capable.

[00149.1.15.15] randomly, with Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the nucleotide sequence of one of nucleotide sequence hybridization shown in 559 row and/or 560-563 are capable has another or various active, described activity for as Table II A or IIB the 3rd be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, protein shown in 559 row and/or 560-563 are capable is known activity or is used for these protein of note.

[0150.0.15.15] in addition, nucleic acid molecule of the present invention can only contain Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the part of the coding region of one of nucleotide sequence shown in 559 row and/or 560-563 are capable, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly has aforementioned activity, for example when its active raising, give margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and described nucleotides sequence column region is listed as with Table I A or IB the 5th or 7 under stringent condition, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the sense strand of one of sequence shown in 559 row and/or 560-563 are capable, Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the antisense sequences of one of sequence shown in 559 row and/or 560-563 are capable or its be natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotide hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the right PCR of primer will produce as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 go and/or 560-563 capable shown in the fragment of polynucleotide sequence.

[0151.0.0.15] sees [0151.0.0.0]

[0152.0.15.15] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and are listed as Table II A or IB the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the abundant homology of aminoacid sequence shown in 559 row and/or 560-563 are capable, make this albumen or its part keep the ability that participates in producing each fine chemicals, specifically be included in plant or the microorganism as described above or raising amino acid as be shown in the examples, margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 particularly, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the activity of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) level.

[0153.0.15.15] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprises and is listed as Table I A or IB the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the amino-acid residue that the aminoacid sequence minimal number was identical or of equal value shown in 559 row and/or 560-563 were capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in protein or its part have Table II A for example as herein described or IIB the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the activity of polypeptide.

[0154.0.15.15] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the complete amino acid sequence homology was at least about 30% shown in 559 row and/or 560-563 were capable, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% also most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.15] to [0156.0.0.15]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.15.15] the present invention relates in addition owing to the genetic code degeneracy is different from Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, one of nucleotide sequence (with its part) shown in 559 row and/or 560-563 are capable and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example giving each fine chemicals increase in the biology) is for example by being listed as Table IV the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the polypeptide of sequence encoding shown in 559 row and/or 560-563 are capable or as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the nucleic acid molecule of polypeptide or its function homologue shown in 559 row and/or 560-563 are capable.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as Table IV the 5th or 7 row, be respectively 174-177 capable and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the consensus sequence of 560-563 shown in capable or as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide or its function homologue shown in 559 row and/or 560-563 are capable.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 5th or 7 row, be respectively capable and/or the 179th row of 174-177 and/or the 180th row and/or the consensus sequence of 181-185 shown in capable or as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet, in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I A or IB the 5th or 7 row, is respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 is capable, preferred Table I A the 5th or 7 row, be respectively capable and/or the 558th, 559 row of 181-185 and/or the 560-563 sequence shown in capable.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 5th or 7 row, 181-185 is capable and/or the 558th, 559 the row and/or 560-563 capable shown in nucleic acid molecule identical.

[0158.0.0.15] to [0160.0.0.15]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.15.15] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.15] sees [0162.0.0.0]

[0163.0.15.15] preferably, under stringent condition with Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.15] sees [0164.0.0.0]

[0165.0.15.15] for example can (for example be listed as Table I A or IB the 5th or 7 in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable) Nucleotide that produces the amino acid replacement that causes " nonessential " amino-acid residue place in is replaced.

[0166.0.0.15] to [0167.0.0.15] sees that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.15.15] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 is capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contains and is listed as Table II A or IIB the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the aminoacid sequence of 560-563 shown in capable be at least about 50% identical aminoacid sequence, and can participate in increasing the generation of each fine chemicals after improving its activity (for example it is expressed).Preferably, the protein of this nucleic acid molecule encoding with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable is identical at least about 60%, more preferably with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, one of sequence shown in 559 row and/or 560-563 are capable is identical at least about 70%, even more preferably with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.15] to [0172.0.0.15] sees that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.15.15] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:13930 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:13930 sequence before use.

[0174.0.0.15] sees [0174.0.0.0]

[0175.0.15.15] for example, the sequence that has 80% homology at protein level and SEQ ID NO:13931 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:13931 sequence.

[0176.0.15.15] is by replacing, insert or lack from being listed as Table II A or IIB the 5th or 7 according to of the present invention, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the functional equivalent that one of polypeptide obtained shown in 559 row and/or 560-563 were capable be listed as according to of the present invention as Table II A or IIB the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, one of polypeptide had at least 30% shown in 559 row and/or 560-563 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide shown in 559 row and/or 560-563 are capable has essentially identical character and discerns.

[0177.0.15.15] is by replacing, insert or lack from being listed as Table I A or IB the 5th or 7 according to of the present invention, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the functional equivalent that obtains of nucleotide sequence shown in 559 row and/or 560-563 are capable be listed as according to of the present invention as Table II A or IIB the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, one of polypeptide had at least 30% shown in 559 row and/or 560-563 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the polypeptide of the essentially identical character of polypeptide shown in 559 row and/or 560-563 are capable.

[0178.0.0.15] sees [0178.0.0.0]

[0179.0.15.15] can (particularly be listed as Table I A or IB the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable) the middle replacement of introducing one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II A or IIB the 5th or 7 and be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the nucleic acid molecule of the homologue of protein sequence shown in 559 row and/or 560-563 are capable.Can by standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 introduce sudden change in the encoding sequence of sequence shown in capable.

[0180.0.0.15] to [0183.0.0.15] sees that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.15.15] is employed to have as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the nucleotide sequence homologous compound of sequence shown in 559 row and/or 560-563 are capable, perhaps come Table II A or IIB the 5th or 7 row freely, 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the homologous compound of nucleotide sequence of sequence shown in 559 row and/or 560-563 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I A or IB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 the row and/or 560-563 capable shown in sequence or from the deutero-nucleotide sequence) by the disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.15.15] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 is capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the 560-563 arbitrary sequence shown in capable in other nucleotide sequence of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 is capable, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the 560-563 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.15.15] also preferred the inventive method comprise as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide of sequence shown in 559 row and/or 560-563 are capable.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 is capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence identical.

[0187.0.15.15] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide of sequence shown in 559 row and/or 560-563 are capable also contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 is capable, preferred Table II B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the encoding sequence of sequence identical.

[0188.0.15.15] still has the polypeptide (=protein) of giving the basic biology of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are capable, preferred Table II A or IIB the 3rd and 5 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, shown in 559 row and/or 560-563 are capable and under the same conditions the polypeptide expressed activity is compared and basic is not reduced.In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence or by its homologue of forming.

[0189.0.15.15] is as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the homologous compound of sequence shown in 559 row and/or 560-563 are capable, or deutero-such as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the homologue of the sequence shown in 559 row and/or 560-563 are capable also refers to truncated sequence, cDNA, the single stranded DNA or the RNA of coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.15] to [0203.0.0.15] sees that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.15.15] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding is being listed as Table II A or IIB the 5th or 7 of mature form at least, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide or its segmental nucleic acid molecule shown in 559 row and/or 560-563 are capable, described nucleic acid molecule is given each fine chemicals in biological or its part, it is margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity increase;

(b) comprise, preferably comprise mature form at least as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the nucleic acid molecule that primer shown in 559 row and/or 560-563 are capable or primer obtain amplification from cDNA library or genomic library, and in biological or its part, give each fine chemicals, it is margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the increase of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like Table IV the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, consensus sequence shown in 559 row and/or 560-563 are capable is also given each fine chemicals in biological or its part, it is margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, it is margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity, described peptide coding such as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the structural domain of the polypeptide shown in 559 row and/or 560-563 are capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I A or IB the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, nucleic acid molecule shown in 559 row and/or 560-563 are capable or coding (optimized encoding is mature form at least) are as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, at least the 15nt of the nucleic acid molecule of polypeptide shown in 559 row and/or 560-563 are capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Thereby the nucleic acid molecule of preferred (a) to (l) is different from as Table I A the 5th or 7 row, is respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the 560-563 sequence shown in capable by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention not by as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence form.In another embodiment, nucleic acid molecule of the present invention with as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In other embodiments, nucleic acid molecule do not encode as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in peptide sequence.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in polypeptide have the different polypeptide of one or more amino acid at least.In another embodiment, as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in nucleic acid molecule do not encode as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the protein of sequence.Therefore, in one embodiment, by nucleotide sequence (a)-(l) encoded protein matter not by Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, protein sequence at least 30% shown in 559 row and/or 560-563 are capable identical and with Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence was less than 100% shown in 559 row and/or 560-563 were capable, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% is identical.

[0205.0.0.15] to [0206.0.0.15]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.15.15] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene, triacylglycerol or lipid, preferably glycerine phosphatide, sphingolipid and/or galactolipid biosynthesis gene or its combination of fatty acid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.15] to [0226.0.0.15] sees that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.15.15] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the capable sequence or derivatives thereof of mentioning of 560-563, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one fatty acid biosynthetic pathway, for example palmitinic acid, stearic acid, Zoomeric acid, oleic acid, linolic acid and/or linolenic acid or triacylglycerol or lipid preferably glycerine phosphatide sphingolipid and/or galactolipid biosynthetic pathway gene in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.15.15] therefore cultivated to cross simultaneously in another embodiment of the present invention and expressed coding and fatty acid metabolism (particularly lipid acid is synthetic) at least one nucleic acid of related protein or the biology of a gene.

[0229.0.15.15] can be the coding saturated fatty acid with other favourable nucleotide sequences that sequence that present method is used and/or the combination of aforementioned biosynthesis gene are expressed; the sequence of other gene of polyunsaturated fatty acid biosynthetic pathway; for example as WO 98/46765; WO 98/46763; WO98/46764; WO 99/64616; WO 00/20603; WO 00/20602; WO 00/40705; US 20040172682; US 20020156254; US 6; 677,145; desaturase such as the Δ-4-desaturase described among US 20040053379 or the US 20030101486; Δ-5-desaturase; Δ-6-desaturase; Δ-8-desaturase; Δ-9-desaturase; Δ-12-desaturase; Δ-17-desaturase; ω-3-desaturase; prolong enzyme such as Δ-5-and prolong enzyme; Δ-6-prolongs enzyme; Δ-9-prolongs enzyme; acyl group-CoA-desaturase; acyl-acp-desaturase; acyl-acp-thioesterase; the fatty acid acyl based transferase; acyl group-CoA lysophospholipid acyltransferase; acyl group-CoA carboxylase; Fatty acid synthetase; fatty acid hydroxylase; acyl group-CoA oxydase; acetylenase; lipoxygenase; three acyl group lipase etc.These genes cause the synthetic increase of indispensable fatty acid.

[0229.1.15.15] can be the coding saturated fatty acid with other nucleotide sequences that sequence that present method is used and/or the combination of aforementioned biosynthesis gene are expressed, unsaturated fatty acids, the sequence of other gene of polyunsaturated fatty acid and/or hydroxylation fatty acid biosynthetic pathway, FA2H gene (the Alderson etc. of lipid acid 2-hydroxylase for example encode, J.Biol.Chem. the 279th roll up, the 47th phase, on November 19th, 2004, the 48562-48568 page or leaf), the FAD2 and the FAD3 (US 20030221217) of coding ω-3-desaturase, Δ-12 fatty acid desaturase, Δ-15 fatty acid desaturase is (at Okuley etc., Plant Cell 6:147-158 (1994); Lightner etc., WO94/11516; YadavN. etc., Plant Physiol., 103:467-476 (1993), WO 93/11245 and Arondel, V. etc., Science, 258:1353-1355 (1992), open among the US 20040083503).

[0230.0.0.15] sees [0230.0.0.0]

[0231.0.15.15] is in another advantageous embodiment of the inventive method, the employed biology of present method is to have weakened degraded margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 simultaneously, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the protein of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid), those biologies of the expression rate by reducing corresponding gene especially.

[0232.0.0.15] to [0276.0.0.15] sees that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.15.15] can separate the lipid acid that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.The fine chemicals that produces according to the inventive method can be as previously mentioned from biological, advantageously from plant with the isolated in form of oils, fat, lipid and/or free fatty acids.The lipid acid of producing by this process can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing or extract realization with plant part, preferred plant seed.Preferred hexane wherein can separate more than 96% of compound that process of the present invention produces as the solvent in this process.Afterwards, resulting product is further processed, promptly come unstuck, make with extra care, bleaching and/or deodorizing.

[0278.0.0.15] to [0282.0.0.15] see[0278.0.0.0] to [0282.0.0.0]

[0283.0.15.15] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-as Table II A or IIB the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, proteinic antibody shown in 559 row and/or 560-563 are capable, for example anti-as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the antibody of polypeptide shown in 559 row and/or 560-563 are capable, it can comprise above-mentioned sequence or by its polypeptide of forming by the standard technique utilization, and polypeptide for example of the present invention or its fragment produce.Monoclonal antibody preferably.

[0284.0.0.15] sees [0284.0.0.0]

[0285.0.15.15] in one embodiment, the present invention relates to have as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence or by as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.15.15] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, consensus sequence shown in 559 row and/or 560-563 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, consensus sequence or by its polypeptide of forming shown in 559 row and/or 560-563 are capable, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the polypeptide of consensus sequence (each row).

[0287.0.0.15] to [0290.0.0.15] sees that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.15.15] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as Table II A or IIB the 5th or 7 row, is respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the 560-563 sequence shown in capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence form.

[0292.0.0.15] sees [0292.0.0.0]

[0293.0.15.15] the present invention relates to give that each fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB, be respectively 174-177 capable and/or the 178th the row and/or the 179th the row and/or the 180th the row and/or 181-185 is capable and/or the 558th, 559 the row and/or 560-563 capable shown in other sequence of sequence phase region.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in sequence form.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.15.15] in one embodiment, the present invention relates to have as Table II the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the polypeptide of activity of proteins shown in 559 row and/or 560-563 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, sequence shown in 559 row and/or 560-563 are capable.

[0295.0.0.15] to [0297.0.0.15] sees that [0295.0.0.0] is to [0297.0.0.0]

The chemical of [00297.1.15.15] non-polypeptide of the present invention be for example do not have Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the active polypeptide of polypeptide.

[0298.0.15.15] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence so that protein or its part have kept giving the present invention active ability.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the identical aminoacid sequence of sequence.

[0299.0.15.15] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table II A or IIB the 5th or 7 be listed as, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the homology of one of aminoacid sequence sequence shown in 559 row and/or 560-563 are capable is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.15.15] therefore, described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 go and/or 560-563 capable shown in sequence different.Therefore, this polypeptide contains and is listed as Table II A or IIB the 5th or 7, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the complete amino acid sequence homology was at least about 35% shown in 559 row and/or 560-563 were capable, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.15] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.15.15] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the aminoacid sequence shown in 559 row and/or 560-563 are capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.15] sees [0303.0.0.0]

[0304.0.15.15] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as Table II A or IIB the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.15] to [0308.0.0.15] sees that [0305.0.0.0] is to [0308.0.0.0]

[0309.0.15.15] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, protein shown in 559 row and/or 560-563 are capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, and be not shown in Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the polypeptide that " non-polypeptide of the present invention " or " other polypeptide " during 559 row and/or 560-563 are capable is meant the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide shown in 559 row and/or 560-563 are capable is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II A or IIB the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 are proteinic and from the protein of identical or different biology shown in capable.In one embodiment, be not shown in Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or capable " the non-polypeptide of the present invention " or " other polypeptide " of 560-563 are not given in biology or its part each fine chemicals and increased.

[0310.0.0.15] to [0334.0.0.15] sees that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.15.15] confirmed the dsRNAi method to reduce as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the expression of the 558th, 559 row and/or the 560-563 nucleotide sequence shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.15] to [0342.0.0.15] sees that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.15.15] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown between the genetic transcription thing of one of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or 560-563 capable shown in one of sequence or its homologue dsRNA of beginning to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.15] to [0361.0.0.15] sees that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.15.15] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide shown in 559 row and/or 560-563 are capable) nucleic acid molecule.

Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.In one embodiment, have as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the transgenosis of the polypeptide of polypeptide active was meant in the text because genomic regulation and control or manipulation shown in 559 row and/or 560-563 were capable, in cell or biological or its part, be noted as Table II A or IIB the 3rd row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide shown in 559 row and/or 560-563 are capable (for example has as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, polypeptide of sequence shown in 559 row and/or 560-563 are capable) activity is enhanced.Example and the inventive method are described in above.

[0363.0.0.15] sees [0363.0.0.0]

[0364.0.15.15] naturally occurring expression cassette---for example naturally occurring coding such as Table II A or IIB the 3rd and/or 5 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the promotor of polypeptide of the present invention shown in 559 row and/or 560-563 are capable is listed as Table I A or IB the 3rd and/or 5 with corresponding, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the combination of protein coding sequence shown in 559 row and/or 560-563 are capable---become transgene expression cassette.

[0365.0.0.15] to [0373.0.0.15] see[0365.0.0.0] to [0373.0.0.0]

[0374.0.15.15] contains in the methods of the invention the transgenic plant of institute's synthetic fatty acid and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the lipid acid that produces in the inventive method also can be from plant with free fatty acids or be bonded to the isolated in form of compound.The lipid acid that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.15] to [0376.0.0.15] sees that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.15.15] therefore the invention still further relates to the method according to this invention, separated the lipid acid and/or fatty acid composition or the fine chemicals that are produced thus.

[0378.0.15.15] can separate by this way and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than 90% the lipid acid that produces in the method.As required, resulting lipid acid can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.15.15] in one embodiment, amino acid, particularly margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) be the mixture that comprises one or more each fine chemicals.In one embodiment, each fine chemicals here looks like and is meant amino acid, particularly margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid).In one embodiment, amino acid here looks like and is meant the mixture of each fine chemicals.

The suitable synthetic parent material of the lipid acid that [0380.0.15.15] obtains in the method for the invention as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises fatty acid composition that separation produces or fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is lipid acid or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine that the inventive method produces.

The lipid acid that [0380.1.15.15] obtains in the method for the invention is also suitable to be used as the quantitative internal standard of lipid acid with purified form.They can also be by prolonging, being used as initial compounds in saturated or desaturation, the synthetic other lipid acid of hydroxylation or synthetic glyceride or lipid, preferably glycerine phosphatide, sphingolipid and/or the galactolipid.

[0381.0.0.15] to [0382.0.0.15] sees that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.15.15] is in order to prepare the fine chemicals of fatty acids compound, fine chemicals particularly, can use organic compound, the oils, fat and/or the lipid that for example contain lipid acid (lipid acid that for example has C10--C16 carbon atom main chain) as precursor compound and/or little organic acid (for example acetate, propionic acid or butyric acid) are as fatty acid source.

[0384.0.0.15] sees [0384.0.0.0]

The fermented liquid that [0385.0.15.15] obtains with this kind approach, particularly contain and other lipid, fat and/or oils blended margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the fermented liquid of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid), dry matter content accounts for 7.5 to 25% of weight usually.At least when finishing but particularly passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means that the concentration that can utilize sugar in the fermention medium during this period of time remains on or be reduced to 0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant) or these methods or partly from fermented liquid, shift out or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.15.15] yet, can also be further purified the lipid acid of generation.For this reason, the composition that will contain product carries out thin-layer chromatography or carries out Florisil column chromatography (BouhoursJ.F., J.Chromatrogr.1979,169,462) on silica-gel plate, and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.An alternative approach of purification of fatty acid is for for example carrying out crystallization under the urea existence condition.These methods can combination with one another.

[0387.0.0.15] to [0392.0.0.15] sees that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.15.15] the present invention relates to identify and gives the method that the compound that becomes more meticulous in the cell produces the gene product that increases that it comprises following step in one embodiment:

(a) sample that will comprise candidate gene (be coded in and give the gene product that fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I A or IB, preferred Table I B the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the 560-563 nucleic acid molecule shown in capable is hybridized nucleic acid molecule under lax stringent condition, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.15] to [0398.0.0.15] sees that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.15.15] in one embodiment, the present invention relates to identify the method for giving the compound that the fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under the fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving fine chemicals generation raising under these conditions.

It is contemplated that by for example seeking the resistance of the medicine of blocking-up lipogenesis and observing this effect and whether depend on YDR513W for example relatively have low and the active biology much at one of high YDR513W, the fine chemicals that screens raising produces.

Whether [00399.1.15.15] it is contemplated that by for example seeking the resistance of blocking-up fine chemicals synthetic medicine and observing this effect and depends on as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, active or the expression of the polypeptide shown in 559 row and/or 560-563 are capable or its homologue, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, the biology phenotype more much at one of activity of proteins shown in 559 row and/or 560-563 are capable.

[0400.0.0.15] to [0416.0.0.15] sees that [0400.0.0.0] is to [0416.0.0.0]

[0417.0.15.15] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of fatty acid biosynthetic pathway is had resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block lipid acid in the described biology (particularly fine chemicals) synthetic inhibitor.The example of lipid acid synthetic inhibitor or weedicide is for suppressing the cerulenin of lipid acid (beta-keto acyl base thioesters synthetase inhibitors) in the blocking-up biological (for example microorganism or plant); Thiolactomycin; Diazoborines or trichlorophenol and sterol biosynthesis inhibitor be chloroformate grass oxazole diclofop-methyl for example; the spirit of pyrrole fluorine chlorine standing grain; fluazifop or quizalofop; the cyclohexyl diketone that perhaps suppresses plant acetyl coenzyme A carboxylase is clethodim or sethoxydim [(2-[1-{ ethoxy imino } butyl]-5-[2-{ ethylmercapto group for example } propyl group]-3-hydroxyl-2-tetrahydrobenzene-1-ketone), perhaps thiocarbamate butylate for example; EPTC[=S-ethyl dipropyl thiol carbamate] or vernolate.

[0418.0.0.15] to [0423.0.0.15] sees that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.15.15] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, agonist with the inventive method evaluation, the nucleic acid molecule of identifying with the inventive method can be used to produce fine chemicals or produce fine chemicals and one or more other lipid acid, margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 particularly, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the mixture of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) mixture or other lipid acid.

Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing the fine chemicals of biological or its part (as cell).

[0425.0.0.15] to [0430.0.0.15] sees that [0425.0.0.0] is to [0430.0.0.0]

[0431.0.15.15] embodiment 1: clone SEQ ID NO:13930 in intestinal bacteria

[0432.0.15.15] uses the method for known good foundation (to consult as Sambrook, J. etc. (1989) " Molecular Cloning:A Laboratory Manual " Cold Spring HarborLaboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Mole-cular Biology ", John Wiley ﹠amp; Sons), with SEQ ID NO:13930 be cloned into plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl Acad.Sci.USA, 75:3737-3741); PACYC177 (Change ﹠amp; Cohen (1978) J.Bacteriol.134:1141-1 156); PBS series plasmid (pBSSK+, pBSSK-and other; Stratagene, LaJolla, USA) or clay such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J.Rosenthal, A., and Waterson, R.H. (1987) Gene 53:283-286) with at expression in escherichia coli.

[0433.0.0.15] to [0434.0.0.15] sees that [0433.0.0.0] is to [0434.0.0.0]

[0435.0.15.15] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.15.15] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement, the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce xenthophylls.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coliand Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces the expection fine chemicals by screening can screen positive catastrophic event.

[0437.0.15.15] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.15.15] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.15.15] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.15.15] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci USA. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.15] sees [0441.0.0.0]

Suddenly change in [0442.0.15.15] observation transformed host cell or transgenic protein activity reaches this fact in the mode similar to wild-type protein with similar scale based on protein.Measuring appropriate method that mutant gene or transgenosis transcribe quantity (can for the sign of the mRNA quantity of translation gene product) (consults as Ausubel etc. for carrying out the Northern trace, (1988) " Current Protocolsin Molecular Biology ", Wiley:New York), wherein provide primer to design with goal gene bonded mode with detectable mark (being generally radioactivity or chemiluminescent labeling), thereby as the total RNA that extracts the biological culture thing, in glue, separate, be applied to stable matrix and when hatching, the combination of probe and indicate the existence and the quantity of this gene mRNA in conjunction with quantity with this probe.Other method is a quantitative PCR.This information detects the degree of genetic transcription.Can use several different methods known in the art, for example use the Ambion test kit and according to the specification sheets of manufacturers, perhaps as Edgington etc., Promega Notes Magazine Number 41,1993, the 14 pages described separates total cell RNA from for example yeast or intestinal bacteria.

[0443.0.0.15] sees [0443.0.0.0]

[0444.0.15.15] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.15.15] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be in pH6.5,20 ℃ of cultivations down in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.15] to [0450.0.0.15] sees that [0446.0.0.0] is to [0450.0.0.0]

[0451.0.15.15] if detect the clone of genetic modification, also should comprise the contrast clone that contains the basic plasmid that has or not insertion or the contrast clone of unmodified in the test.If the express transgenic sequence also should advantageously comprise the contrast clone in the test.Use is at 30 ℃ of agar plates of hatching, as cultured cells on the CM flat board (the poly-peptone of 10g/l glucose, 2.5g/l NaCl, 2g/l urea, 10g/l, 5g/l yeast extract, 5g/l meat extract, 22g/l agar transfer to pH value 6.8 with 2M NaOH) advantageously substratum is hatched to OD600 be 0.5 to 1.5.For example come inoculation medium by adding the pre-incubated seed biology of liquid (for example intestinal bacteria or yeast saccharomyces cerevisiae).

[0452.0.0.15] to [0453.0.0.15] sees that [0452.0.0.0] is to [0453.0.0.0]

[0454.0.15.15] embodiment 8: the influence that analyzing nucleic acid molecules produces lipid acid

[0455.0.15.15] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example lipid acid) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being lipid or lipid acid) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

Except said process, as (1999) Proc.Natl.Acad.Sci.USA 96 (22) such as Cahoon: from vegetable material, extract plant lipid as described in (1986) Analytic Biochemistry 152:141-145 such as 12935-12940 and Browse.The qualitative and quantitative analysis of lipid or lipid acid is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:Oily Press (Oily Press Lipid Library; 2); Christie, William W., GasChromatography and Lipids.A Practical Guide-Ayr, Scotland:Oily Press, 1989, Repr.1992, IX, 307 pp. (Oily Press Lipid Library; 1); " Progress inLipid Research, Oxford:Pergamon Press, 1 (1952)-16 (1977), exercise question is Progress in the Chemistry of Fats and Other Lipids CODEN.

[0456.0.0.15] sees [0456.0.0.0] except measuring the fermentation end product, also can analyze other components (as intermediate product and by product) of the pathways metabolism that is used for producing described compound, with the yield of measuring biological total output, compound and/or produce efficient.Analytical procedure comprises amount (for example sugar, carbohydrate, nitrogenous source, phosphoric acid salt and other ions), the mensuration biomass composition of measuring nutrient substance in the substratum and grows, analyzes the generation of biosynthetic pathway eubolism thing and measure the gas that produces between yeast phase.These standard methods are described in " Applied Microbial Physiology; APractical Approach ", P.M.Rhodes and P.F.Stanbury compile IRL Press, 103-129 page or leaf; 131-163 page or leaf and 165-192 page or leaf (ISBN:0199635773) and the reference of quoting thereof.

[0457.0.15.15] embodiment 9: the purifying of lipid acid

[0458.0.15.15] embodiment analyzes lipid acid (abbreviation: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatograph/mass spectrum; TAG, triacylglycerol; TLC, thin-layer chromatography).By using standard method of analysis GC, GC-MS or TLC can clearly detect the existence of fatty acids products to the biology analysis of recombinating, described analytical procedure Christie and reference wherein are described (1997, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric method], Lipide33:343-353).The biological total fatty acids that yeast produced that for example is used for the inventive method can be according to the following step analysis: by ultrasonic, the grinding of glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed such as yeast, intestinal bacteria or plant fragmentation.After the fragmentation, must material is centrifugal (1000xg, 10 minutes, 4 ℃) also use 100mM NaHCO ₃, pH 8.0 washings are once to remove residual substratum and lipid acid.In order to prepare fatty acid methyl ester (FAMES), with pellet resuspended in distilled water, in 100 ℃ of heating 10 minutes, cooled on ice is also centrifugal, then in containing the 0.5M sulfuric acid (in methyl alcohol) of 2% Propanal dimethyl acetal, extracted 1 hour in 90 ℃, this causes producing the oil and the fat compound of hydrolysis, and this can obtain transmethylase fat.Then, use 2ml Petroleum ether extraction FAMES twice, use 100mM NaHCO then ₃, pH8.0 washs once and uses Na ₂SO ₄Dry.Under argon gas stream,, and FAMES heavily is dissolved in the 50 μ l sherwood oils organic solvent evaporation.ZEBRON ZB-Wax capillary column in HewlettPackard 6850 gas chromatographs that have flame ionization detector (30m, 0,32mm, 0,25 μ m; Phenomenex) go up sample separation.Furnace temperature is carried out time variable control, with 20 ℃/minute speed from 70 ℃ (keeping 1 minute) to 200 ℃, then with 5 ℃/minute speed to 250 ℃ (keeping 5 minutes), at last with 5 ℃/minute speed to 260 ℃.Nitrogen is as carrier gas (4.5ml/ minute, under 70 ℃).The identity of resulting fatty acid methyl ester must be used and can define from the standard that commercial source (being Sigma) obtains.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Then, in 100 ℃ of heating 10 minutes, centrifugation again after the cooled on ice.With cell precipitation in 1M sulfuric acid methanol solution and 2% Propanal dimethyl acetal in 90 ℃ of hydrolysis 1 hour, lipid is by transmethylase.Resulting fatty acid methyl ester (FAME) extracts in sherwood oil.The FAME that is extracted by use capillary column (Chrompack, the WCOT fused silica, CP-Wax-52CB, 25 μ m 0.32mm) carried out gas chromatographic analysis in following 5 minutes in 170 ℃-240 ℃ gradient temperature 20 minutes and 240 ℃.The identity of fatty acid methyl ester must be by relatively confirming with corresponding FAME (being Sigma).Can be suitable chemically derived by the FAME mixture is carried out, for example obtain 4 by GC-MS, 4-Er Jia Yang oxazolin derivs (Christie, 1998) is further identified identity and position of double bond.

Method is described in as Napier and Michaelson, and 2001, Lipids.36 (8): 761-766; Sayanova etc., 2001, Journal of Experimental Botany.52 (360): 1581-1585, Sperling etc., 2001, Arch.Biochem.Biophys.388 (2): 293-298 and Michaelson etc., 1998, FEBS Letters.439 (3): among the 215-218.

[0459.0.15.15] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified lipid acid.Because of the elutriant difference, can advantageously use acetonitrile/water and chloroform/acetonitrile mixture.For example, by using RP-18-post (ET 250/3Nucleosil 120-5C ₁₈Macherey und Nagel, D ü ren, this HPLC method Germany) has been separated rapeseed oil.Chloroform/acetonitrile mixture (v/v 30:70) is as elutriant.0.8ml/ minute flow velocity be favourable.In order to analyze lipid acid, used ELSD detector (light scattering detector).For the purifying of lipid acid, MPLC, dried post flash chromatography (dry-flash chromatography) or thin-layer chromatography are other favourable chromatographic processes.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.15.15] depends on the fine chemicals that is produced in addition, can also use crystallization and distillation to come purifying.These two kinds of methods are that those skilled in the art are well-known.

[0461.0.15.15] embodiment 10: clone SEQ ID NO:13930 is used for expressing plant

[0462.0.0.15] sees [0462.0.0.0]

[0463.0.15.15] passes through pcr amplification SEQ ID NO:13930 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

The composition of [0464.0.15.15] Pfu Turbo archaeal dna polymerase schedule of operation is as follows: 1x PCR damping fluid (Stratagene), every kind of dNTP of 0.2mM, 100ng yeast saccharomyces cerevisiae (bacterial strain S288C; Research Genetics, Inc. now is Invitrogen) or intestinal bacteria (bacterial strain MG1655; E.coli Genetic Stock Center) genomic dna, 50pmol forward primer, 50pmol reverse primer, 2.5u Pfu Turbo archaeal dna polymerase.Amplification cycles is as follows:

[0465.0.15.15] 94-95 ℃ of circulation in 3 minutes, 95 ℃ of 1 minute or 94 ℃ 30 seconds, 50 ℃ 45 seconds, 50 ℃ 30 seconds or 55 ℃ 30 seconds and 72 ℃ of 210-480 seconds then, 28-36 circulation under each situation, 72 ℃ of circulations in 8 minutes then, 4 ℃ then.

[0466.0.0.15] sees [0466.0.0.0]

[0467.0.15.15] selects following primer sequence for genes of SEQ ID NO:13930:

I) forward primer (SEQ ID NO:13950)

atgaactcta?ttttagacag?aaatgtt

Ii) reverse primer (SEQ ID NO:13951)

ttatttttgg?tcttgtttca?aagtgtc

Perhaps as Table III the 7th row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the 560-563 primer shown in capable be used for selected at Table III the 5th row, 174-177 is capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the 558th, 559 row and/or the capable described genes of 560-563.

[0468.0.0.15] to [0479.0.0.15] sees that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.15.15] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:13930

[0481.0.0.15] to [0513.0.0.15] sees that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.15.15] selects as another kind of, as people such as Geigenberger (Plant Cell ﹠amp; Environ, 19,1996:43-55) described, can be separated in by HPLC and advantageously detect lipid acid in the ethanol extraction.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??YDR513W	Margaric acid (C17:0)	??GC	??1.24	??1.97
??YER156C	Margaric acid (C17:0)	??GC	??1.20	??1.49
					??YLR255C	Margaric acid (C17:0)	??GC	??1.20	??1.27
??b1829	Margaric acid (C17:0)	??GC	??1.20	??2.33
					??YER173W	The 2-hydroxy-palmitic acid	??GC	??1.26	??4.94
??YFR042W	2-hydroxyl tetracosenoic acid	??GC	??1.28	??1.56
					??YOR317W	Hexadecadienoic acid	??GC	??1.19	??1.69
??YGL205W	Hiragonic acid (C16:3)	??GC	??1.14	??1.16
					??YIL150C	Hiragonic acid (C16:3)	??GC	??3.24	??3.24
??YKL132C	Hiragonic acid (C16:3)	??GC	??1.13	??1.56
					??YOR344C	Hiragonic acid (C16:3)	??GC	??1.12	??1.20
??b3430??b0057	Hiragonic acid (C16:3) C24:1 lipid acid	??GC??GC	??1.12??1.22	??1.20??1.37
					??b0161	The 2-hydroxy-palmitic acid	??GC	??1.21	??1.48
??b0758	The 2-hydroxy-palmitic acid	??GC	??1.19	??1.38
					??b1097	C24:1 lipid acid	??GC	??1.23	??1.41
??b2078	C24:1 lipid acid	??GC	??1.23	??1.41
					??b3231	C24:1 lipid acid	??GC	??1.23	??1.48

[0515.0.15.15] the 2nd row have shown the lipid acid of being analyzed.The 4th row and the 5th row have shown the ratio of lipid acid between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

[0516.0.0.15] sees [0516.0.0.0]

[0517.0.15.15] embodiment 14a: transform the rye grass plant by yeast saccharomyces cerevisiae YOR344C or from the expression of crossing of other biological YOR344C homologue

[0518.0.0.15] to [0524.0.0.15] sees that [0518.0.0.0] is to [0524.0.0.0]

[0525.0.15.15] embodiment 14b: express the soybean transformation plant by yeast saccharomyces cerevisiae YOR344C or from crossing of other biological YOR344C homologue

[0526.0.0.15] to [0529.0.0.15] sees that [0526.0.0.0] is to [0529.0.0.0]

[0530.0.15.15] embodiment 14c: transform maize plant by yeast saccharomyces cerevisiae YOR344C or from the expression of crossing of other biological YOR344C homologue

[0531.0.0.15] to [0533.0.0.15] sees that [0531.0.0.0] is to [0533.0.0.0]

[0534.0.15.15] embodiment 14d: transform wheat plant by yeast saccharomyces cerevisiae YOR344C or from the expression of crossing of other biological YOR344C homologue

[0535.0.0.15] to [0537.0.0.15] sees that [0535.0.0.0] is to [0537.0.0.0]

[0538.0.15.15] embodiment 14e: transform vegetable seed/rape plant by yeast saccharomyces cerevisiae YOR344C or from the expression of crossing of other biological YOR344C homologue

[0539.0.0.15] to [0542.0.0.15] sees that [0539.0.0.0] is to [0542.0.0.0]

[0543.0.15.15] embodiment 14f: transform the alfalfa plant by yeast saccharomyces cerevisiae YOR344C or from the expression of crossing of other biological YOR344C homologue

[0544.0.0.15] to [0547.0.0.15] sees that [0544.0.0.0] is to [0547.0.0.0]

[0548.0.15.15] embodiment 14g: transform the alfalfa plant by yeast saccharomyces cerevisiae YOR344C or from the expression of crossing of other biological YOR344C homologue

[0549.0.0.15] to [0552.0.0.15] sees that [0549.0.0.0] is to [0552.0.0.0]

[0552.1.0.15]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

?ORF	Metabolite	??Min	??Max
				?b1829	Margaric acid (C17:0)	??1.65	??1.66

Table 2 shows that margaric acid (C17:0) has increased in the genetic modification maize plant of expressing e. coli k12 nucleotide sequence b1829.

E. coli k12 protein b1829 or have under the situation that the activity that is defined as the active protein of heat shock protein or its homologue is enhanced in maize plant, preferably, giving fine chemicals margaric acid (C17:0) is increased between 65% and 66%.

[00552.2.0.15] sees [00552.2.0.0]

[0553.0.15.15]

1. produce margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid), it comprises:

(a) improve in non-human being or its one or more parts or produce as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or 558-559 and/or 560-563 capable shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, produce margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or under the condition of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) cultivate this biology.

2. produce margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the method for 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid), be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or 558-559 and/or 560-563 capable shown in polypeptide or its fragment, described nucleic acid molecule is given margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in biology or its part, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity increase;

B) contain just like Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the nucleic acid molecule of 558-559 and/or the 560-563 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity;

E) under stringent hybridization condition, hybridize and give margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in biology or its part with nucleic acid molecule (a) to (c), 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity;

F) nucleic acid molecule, it comprise by use as Table III the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or 558-559 and/or 560-563 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively 174-177 capable and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or 558-559 and/or 560-563 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises recovery free or bonded margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid).

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded margaric acid and/or 2-hydroxy-palmitic acid and/or the 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (the preferred Δ 7 of selected mutation biology or the generation of its part, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid).

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is as Table II A or IIB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or 558-559 and/or 560-563 capable shown in polypeptide or its fragment, described nucleic acid molecule is given margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in biology or its part, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity increase;

B) contain just like Table I A or IB the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or the nucleic acid molecule of 558-559 and/or the 560-563 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) nucleic acid molecule of quantity increase;

E) under stringent hybridization condition, hybridize and give margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in biology or its part with nucleic acid molecule (a) to (c), 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity;

F) nucleic acid molecule, it comprise by use as Table III the 7th row, be respectively 174-177 capable and/or the 178th row and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or 558-559 and/or 560-563 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give that fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively 174-177 capable and/or the 179th row and/or the 180th row and/or 181-185 is capable and/or 558-559 and/or 560-563 capable shown in consensus sequence polypeptide and give biology or its part in fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, be respectively 174-177 capable and/or the 178th the row and/or the 179th the row and/or the 180th the row and/or 181-185 is capable and/or 558-563 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding as claim 6 described in, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 row, be respectively 174-177 capable and/or the 178th row and/or the 179th is gone and/or the 180th go and/or 181-185 is capable and/or 558-563 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the agonist of screening polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity increase, described method comprises:

(a) will express coded and give in biology or its part margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 by the nucleic acid molecule of claim 6,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or cell, tissue, plant or the microorganism of the polypeptide that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity and candidate compound or the sample that comprises multiple compound under the condition of this expression of polypeptides of permission, contact;

(b) measure in cell, tissue, plant or the microorganism, perhaps palmitinic acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the palmitinic acid level that will measure or expression of polypeptides level and standard palmitinic acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and give margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in plant or the microorganism, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) produce the method for the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the polypeptide that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and it is coded and give margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in biology or its part to be provided at the nucleic acid molecule that allows described read-out system and claim 6,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or under the condition of the expression of polypeptides of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity increase respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and give margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in the cell, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) produce the method for the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, described sample nucleic acid molecule can contain to be coded in gives margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 after the expression, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the candidate gene of the gene product that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid);

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported suitable margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (the preferred Δ 7 of producing, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the host cell of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid);

(d) in host cell, express the nucleic acid molecule of being identified;

(e) margaric acid in the analysis host cell and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) level and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) level; With

(f) identify nucleic acid molecule and gene product thereof, compare with wild-type, after expressing, it gives margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in the host cell, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) level improve.

20. identify and give margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in the cell, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) produce the method for the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain to be coded in gives margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in biology or its part after the expression, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the candidate gene of the gene product that improves of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) quantity or level, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported suitable margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (the preferred Δ 7 of producing, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the host cell of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid);

(c) in host cell, express the nucleic acid molecule of being identified;

(d) margaric acid in the analysis host living beings and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7,10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) level; With

(e) identify nucleic acid molecule and gene product thereof, compare with wild-type, after expressing, it gives margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 in the host cell, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) level improve.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used for identifying gives margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 after expression, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the nucleic acid molecule that increases of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid).

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying and can gives regulation and control margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (preferred Δ 7 at biology, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or the compound of 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) level.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the nucleic acid molecule of claim 6, the polypeptide of claim 14, the nucleic acid construct of claim 7, claim 8 or 9 carrier, antagonist or agonist according to claim 17 evaluation, the antibody of claim 15, the plant of claim 16 or plant tissue, the results material of claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make anti-margaric acid and/or 2-hydroxy-palmitic acid and/or 2-hydroxyl tetracosenoic acid and/or hexadecadienoic acid (the preferred Δ 7 of suppressing of plant, 10 hexadecadienoic acids) and/or hiragonic acid (preferred Δ 7,10,13 hiragonic acids) and/or 24:1 lipid acid (preferred C24:1 Δ 15 lipid acid) synthetic weedicide.

[0554.0.0.15] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.16] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.16] sees [0001.0.0.0]

[0002.0.16.16] finds citromalic acid (2000 NatureBiotechnology 18 such as Fiehn in mouse ear mustard belongs to, 1157-1161) the metabolic new aspect of (the potential precursor of pyruvic acid and acetate) prompting carbon, there is the tricarboxylic acid cycle bypass of before only finding in prompting in bacterium in addition.

Oxysuccinic acid oxaloacetate shuttle is the feature of vegetable cell.This mechanism is at intracellular transport redox Equivalent.

Oxysuccinic acid is not only the center metabolite in the carbon stream intermediate in the biology.In higher plant, the accumulation of vesica oxysuccinic acid is also changeed the transhipment of vacuole oxysuccinic acid thus and have primary effect in many physiological function.These physiological functions are included in the elongation growth motion, pH regulator of the adjusting of infiltration and potential for gas expansion and pore and pulvinus for example in the nitrate reduction process, and other (is summarized, sees L ü ttge etc., Plant Physiol, 124 (2000), 1335-1348).

Osawa and Matsumoto, Plant Physiol, 126 (2001), 411-420 has discussed the aluminium resistance in the oxysuccinic acid involved in plant.

Oxysuccinic acid is the common moiety of whole plants, and its formation is subjected to being called the control of the enzyme (protein catalyzer) of malate dehydrogenase (malic acid dehydrogenase) (MDH).

Oxysuccinic acid has vital role in plant metabolism.Its importance in the nutrition of plant and mineral matter be by its symbiotic nitrogen fixation, phosphorus obtain with aluminium tolerance in the vital role that risen reflect.

During phosphorus lacked, oxysuccinic acid was usually secreted out so that discharge the phosphorus that can not utilize form from root

In nitrogen-fixing root nodule, thereby oxysuccinic acid is the elementary substrate that is used for bacterial respiratory effect activation nitrogenase.

Pyruvic acid is a naturally occurring composition in plant and vegetables and the body, and pyruvic acid participates in metabolism inherently therein, by the metabolism generate energy.Pyruvic acid is represented the final step in glucose or the starch metabolism.

The known output that in yeast strain, improves pyruvic acid (WO 04/099425).

R-Glyceric acid is particularly important precursor in Serine and the glycine anabolism of amino acid.In addition, the energy level of cell may depend on the R-Glyceric acid level of being found.Glycerate and glycerate-3-phosphoric acid form shuttle so that the transport energy equivalent, for example in the photorespiration process between glycolysis enzyme body and peroxysome.

Succsinic acid is the intermediate that acts on the tricarboxylic acid cycle (and glyoxylate cycle) that succinyl CoA produces by succinyl CoA synthetase.Succsinic acid is converted into fumaric acid by the effect of succinodehydrogenase (following FADH2 to form).

Trihydroxy-butyric acid, three hydroxybutyric acid lactones can be taken as the supposition precursor of pheromone sample metabolite in the plant.

Because citromalic acid, R-Glyceric acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, these favourable physiological roles of three hydroxybutyric acids or trihydroxy-butyric acid and Agricultural biotechnologies potential, need to identify the participation citromalic acid, R-Glyceric acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, the metabolic enzyme of three hydroxybutyric acids or trihydroxy-butyric acid or other proteinic gene, and produce to have and utilize these genetic modification citromalic acids, R-Glyceric acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, the mutant of trihydroxy-butyric acid or trihydroxy-butyric acid content or transgenic plant strain.

[0003.0.0.16]-[0007.0.0.16]-/-

A kind of method that [0008.0.0.16] improves biosynthesizing throughput is to use recombinant DNA technology.Therefore, people are desirably in and produce R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid in the plant.The inhuman production of this type allows the most suitable and the most effective quality, quantity and the screening of producing biology of control.The latter is particularly important for commercial production economics, and also is favourable for the human consumer therefore.In addition, people are desirably in and produce R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid in the plant, so that improve plant production power and to the resistance of biological and abiotic stress as mentioned above.

More already used year of recombinant DNA technology method is with by increasing different biosynthesis genes and study the production that its influence to fine chemicals production improves fine chemicals in microorganism and the plant.For example, it is reported that the xenthophylls astaxanthin can result from the nectary of rotaring gene tobacco plant.Those transgenic plant are to prepare by the carrier transformation of tobacco plant that Agrobacterium tumefaciems mediate, and wherein carrier comprises the ketolase encoding gene (called after crtO) from H.pluvialis, and the Pds gene of tomato is as the promotor and the leader sequence of encoding.Those results show that about 75% carotenoid that is found in the conversion plant flowers contains ketone group.

[0009.0.16.16] if can obtain producing algae, plant or other microorganism of a large amount of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid, should be favourable therefore.The protokaryon or the eukaryotic microorganisms that relate to this type of conversion in some embodiments that the present invention is hereinafter discussed.

If it also is favourable can obtaining root, leaf, stem, fruit or spend the middle plant that produces a large amount of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

The quality that [0010.0.16.16] therefore improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or the trihydroxy-butyric acid that for example are present in as mentioned above in plant and some microorganisms are limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is specific R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid spectrum in the balanced diet as far as possible, because excess of glycerol acid on the specific concentrations, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid have negative effect.Further improving the quality only may be by adding other R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid, and these compositions all are limited.

[0011.0.16.16] is necessary with balance mode R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid to be added into suitable biology in order to guarantee the high quality of food and animal-feed.

[0012.0.16.16] therefore, still be starved of one or more codings and participate in R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or the biosynthetic enzyme of trihydroxy-butyric acid or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but need to improve the method that obtains the highest intrinsic energy R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid forever, on the other hand, reduce the byproduct that produces in the production process as far as possible.

[0013.0.0.16] sees [0013.0.0.0]

[0014.0.16.16] therefore, in first embodiment, the present invention relates to produce the method for fine chemicals, wherein fine chemicals is R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid.Therefore, in the present invention, term " fine chemicals " is meant R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid as used herein.

[0015.0.16.16] in one embodiment, term " fine chemicals " or " each fine chemicals " meaning be meant have R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or the active at least a chemical compound of trihydroxy-butyric acid.

In one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant R-Glyceric acid.In one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant citromalic acid.In one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant fumaric acid.In one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant oxysuccinic acid.In one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant pyruvic acid.In one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant succsinic acid.In one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant trihydroxy-butyric acid.In one embodiment, term " fine chemicals " or " each fine chemicals " meaning are meant three hydroxybutyric acids, and this depends on the context that uses it.In whole specification sheets, term " fine chemicals " or " each fine chemicals " meaning is meant R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid, its salt, ester, thioesters or free form or is bonded to other compound such as the form of sugar or glycopolymers (as glucosides, for example bioside).

Especially, the technician knows that anionic compound such as acid are present in the aqueous solution with the balance between acid and its salt according to the pK value of pH value in each chamber in cell or the organism and acid.Therefore, term " fine chemicals ", term " each fine chemicals ", term " acid " or mention in the title used during with anionic compound relevant with the anionic form and the neutral condition of compound.

Therefore, citromalic acid also relates to citromalic acid salt, fumaric acid also relates to fumarate, oxysuccinic acid also relates to the oxysuccinic acid malate, pyruvic acid also relates to pyruvate salt, succsinic acid relates to succinate, and three hydroxybutyric acids relate to three hydroxybutyric acid salt or trihydroxy-butyric acid also relates to trihydroxy-butyric acid trihydroxy-butyric acid salt.

In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning is meant to have the active at least a chemical compound of above-mentioned fine chemicals.

[0016.0.16.16] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YMR241W, YJL099W, YJL055W, YGR007W, YCR059C, YCL032W, YBR184W, YBR084W, b4139, b1676, b1611, b0695, b0730, b1896, b2699, b4063, YBL015W, YFR007W, YJL072C, YKL132C, YOR044W, YPR024W or YPR138C protein perhaps have as Table I the 5th or 7 row, the protein of the coded peptide sequence of nucleic acid molecule shown in the capable or 564-594 of 190-226 is capable; With

(b) in allowing described biology, produce fine chemicals, for example make biological growth under the condition of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid.

Therefore, the present invention relates to produce the method for fine chemicals, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, be respectively the capable or 564-594 of 190-226 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, be respectively the capable or 564-594 of 190-226 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) allow to produce fine chemicals, for example R-Glyceric acid (referring to that 572-576 is capable), citromalic acid (referring to the 190th and/or 564 row), fumaric acid (referring to that 191-205 and/or 565-571 are capable), oxysuccinic acid (referring to that 206-217 and/or 577-583 are capable), pyruvic acid (referring to the 220th or 584 row), succsinic acid (referring to that 221-224 and/or 585-591 are capable), three hydroxybutyric acids (referring to the 218th or 219 row) and/or trihydroxy-butyric acid (refer to the 225th and 226 and/or 592-594 capable) condition under make biological growth.

[0016.1.16.16] therefore, term " fine chemicals " meaning is meant and Table I the 190th and/or relevant " citromalic acid " or its homologue of the 564 listed full sequences of row.Therefore, term " fine chemicals " meaning is meant " fumaric acid " or its homologue relevant with Table I 191-205 and/or the capable listed sequence of 565-571.Therefore, term " fine chemicals " meaning is meant " oxysuccinic acid " or its homologue relevant with Table I 206-217 and/or the capable listed sequence of 577-583.Therefore, term " fine chemicals " meaning is meant and Table I the 218th or relevant " three hydroxybutyric acids " or its homologue of the 219 listed sequences of row.Therefore, term " fine chemicals " meaning is meant and Table I the 220th or relevant " pyruvic acid " or its homologue of the 584 listed sequences of row.Therefore, term " fine chemicals " meaning is meant " succsinic acid " or its homologue relevant with Table I 221-224 and/or the capable listed sequence of 585-591.Therefore, term " fine chemicals " meaning is meant and Table I the 225th and 226 and/or relevant " trihydroxy-butyric acid " or its homologue of the capable listed sequence of 592-594.Therefore, term " fine chemicals " meaning is meant " R-Glyceric acid " or its homologue relevant with the capable listed sequence of Table I 572-576.

Therefore, according to environment and context, term " fine chemicals " can refer to " R-Glyceric acid ", " citromalic acid ", " fumaric acid ", " oxysuccinic acid ", " three hydroxybutyric acids ", " pyruvic acid ", " succsinic acid " or " trihydroxy-butyric acid ".Be meant and " R-Glyceric acid ", " citromalic acid " or " fumaric acid " or " oxysuccinic acid " or " three hydroxybutyric acids " or " pyruvic acid " or " succsinic acid " or " trihydroxy-butyric acid " from the listed sequence of context can also use term " each fine chemicals " in order to illustrate term " each fine chemicals " meaning.

In one embodiment, the inventive method is given more than one each fine chemicals, i.e. one or more organic acids: " R-Glyceric acid ", " citromalic acid ", " fumaric acid ", " oxysuccinic acid ", " three hydroxybutyric acids ", " pyruvic acid ", " succsinic acid " and/or " the content increase of trihydroxy-butyric acid.

[0017.0.0.16] to [0018.0.0.16]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.16.16] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, the protein of protein active shown in the capable or 564-594 of 190-226 is capable or by as Table I the 5th or 7 row, the coded activity of proteins of nucleic acid molecule was carried out above-mentioned modification shown in the capable or 564-594 of 190-226 was capable.

[0020.0.16.16] is surprisingly found out that, as Table I or II the 4th row, the 571st, 582-583, the listed yeast saccharomyces cerevisiae protein of 591 row, YBL015W for example, YCR059C, YFR007W, YJL055W, YJL099W, YMR241W, YPR024W, YPR138C, YBR084W, YBR184W, YCL032W, YGR007W, YJL072C, YKL132C or YOR044W or as Table I or II the 4th row, 564-570,572-581,584-590, e. coli k12 protein shown in 592-594 is capable, for example b1896, b0730, b1611, b2699, b4139, b1676, b0695 or b4063 transgene expression in Arabidopis thaliana is given transform in the plant and described protein and homologue and coding nucleic acid molecule thereof (particularly as Table II the 3rd row, shown in the capable or 564-594 of 190-226 is capable) relevant R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid (" fine chemicals " or " each fine chemicals ") content increases.

[0021.0.0.16] sees [0021.0.0.0]

The sequence of [0022.0.16.16] e. coli k12 b1343 has been published in Blattner, Science277 (5331), 1453-1474,1997, and its activity to be defined as be the ATP RNA-dependent helicase that is stimulated by 23S rRNA.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary " by the ATP RNA-dependent helicase of 23S rRNA stimulation " or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be trihydroxy-butyric acid and/or the composition that comprises trihydroxy-butyric acid, particularly increase the quantity of the trihydroxy-butyric acid of free or combining form.In one embodiment, have rRNA processing in the methods of the invention or translate active activity of proteins and be enhanced or produce, for example be enhanced or produce from the colibacillary rRNA of having processing or the activity of translating active protein or its homologue.Therefore, in one embodiment, the activity of the ATP RNA-dependent helicase that is stimulated by 23S rRNA or its homologue is enhanced and is used to produce fine chemicals in the methods of the invention, promptly trihydroxy-butyric acid, particularly increase the quantity of the trihydroxy-butyric acid of free or combining form.

The sequence of e. coli k12 b3160 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the monooxygenase with luciferase sample atpase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the monooxygenase with luciferase sample atpase activity of colibacillary supposition or the purposes of its homologue, as described its to be used for producing fine chemicals in biological or its part be succsinic acid, particularly increase succsinic acid and/or comprise the composition of succsinic acid, the preferred quantity of the succsinic acid of free or combining form.In one embodiment, the activity of Jia Ding the monooxygenase with luciferase sample atpase activity is enhanced or produces in the methods of the invention, for example is enhanced or produces from the monooxygenase with luciferase sample atpase activity of colibacillary supposition or the activity of its homologue.

The sequence of e. coli k12 b3231 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L13.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 50S ribosomal subunit protein matter L13 or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be pyruvic acid and/or the composition that comprises pyruvic acid, particularly increase pyruvic acid, the preferred quantity that increases the pyruvic acid of free or combining form.In one embodiment, the activity of 50S ribosomal subunit protein matter L13 is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 50S ribosomal subunit protein matter L13 or its homologue is enhanced or produces.

The sequence of e. coli k12 b3231 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L13.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 50S ribosomal subunit protein matter L13 or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be R-Glyceric acid and/or the composition that comprises R-Glyceric acid, particularly increase R-Glyceric acid, the preferred quantity of the R-Glyceric acid of free or combining form.In one embodiment, the activity of 50S ribosomal subunit protein matter L13 is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 50S ribosomal subunit protein matter L13 or its homologue is enhanced or produces.

The sequence of e. coli k12 b3231 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L13.Therefore, in one embodiment, the inventive method comprises the purposes from intestinal bacteria 50S ribosomal subunit protein matter L13 or its homologue as shown here, it is used for producing pyruvic acid and R-Glyceric acid and/or comprising pyruvic acid and the composition of R-Glyceric acid in biological or its part as described, particularly increases pyruvic acid and R-Glyceric acid, preferably free or the pyruvic acid of combining form and the quantity of R-Glyceric acid.In one embodiment, the activity of 50S ribosomal subunit protein matter L13 is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 50S ribosomal subunit protein matter L13 or its homologue is enhanced or produces.

The sequence of e. coli k12 b1738 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be PEP dependency phosphotransferase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary PEP dependency phosphotransferase or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be succsinic acid and/or the composition that comprises succsinic acid, particularly increase succsinic acid, the preferred quantity of the succsinic acid of free or combining form.In one embodiment, the activity of PEP dependency phosphotransferase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary PEP dependency phosphotransferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b1738 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be PEP dependency phosphotransferase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary PEP dependency phosphotransferase or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and/or the composition that comprises fumaric acid, particularly increase fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, the activity of PEP dependency phosphotransferase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary PEP dependency phosphotransferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b1738 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be PEP dependency phosphotransferase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary PEP dependency phosphotransferase or its homologue as shown here, it is used for producing fumaric acid and succsinic acid and/or comprising fumaric acid and the composition of succsinic acid in biological or its part as described, particularly increases fumaric acid and succsinic acid, preferably free or the fumaric acid of combining form and the quantity of succsinic acid.In one embodiment, the activity of PEP dependency phosphotransferase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary PEP dependency phosphotransferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b0161 (accession number NP_414703) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary pericentral siphon serine protease or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and/or the composition that comprises fumaric acid, particularly increase fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, the activity of pericentral siphon serine protease is enhanced or produces in the methods of the invention, and for example the activity from colibacillary pericentral siphon serine protease or its homologue is enhanced or produces.

The sequence of e. coli k12 b0161 (accession number NP_414703) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary pericentral siphon serine protease or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid and/or the composition that comprises oxysuccinic acid, particularly increase oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, the activity of pericentral siphon serine protease is enhanced or produces in the methods of the invention, and for example the activity from colibacillary pericentral siphon serine protease or its homologue is enhanced or produces.

The sequence of e. coli k12 b0161 (accession number NP_414703) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary pericentral siphon serine protease or its homologue as shown here, it is used for producing oxysuccinic acid and fumaric acid and/or comprising oxysuccinic acid and the composition of fumaric acid in biological or its part as described, particularly increases oxysuccinic acid and fumaric acid, preferably free or the oxysuccinic acid of combining form and the quantity of fumaric acid.In one embodiment, the activity of pericentral siphon serine protease is enhanced or produces in the methods of the invention, and for example the activity from colibacillary pericentral siphon serine protease or its homologue is enhanced or produces.

The sequence of e. coli k12 b1693 (accession number NP_416208) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the 3-dehydroquinate dehydratase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 3-dehydroquinate dehydratase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be R-Glyceric acid and/or the composition that comprises R-Glyceric acid, particularly increase R-Glyceric acid, the preferred quantity of the R-Glyceric acid of free or combining form.In one embodiment, 3-dehydroquinate dehydratase activity of proteins is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 3-dehydroquinate dehydratase protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b1693 (accession number NP_416208) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the 3-dehydroquinate dehydratase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 3-dehydroquinate dehydratase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be citromalic acid and/or the composition that comprises citromalic acid, particularly increase citromalic acid, the preferred quantity of the citromalic acid of free or combining form.In one embodiment, 3-dehydroquinate dehydratase activity of proteins is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 3-dehydroquinate dehydratase protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b1693 (accession number NP_416208) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the 3-dehydroquinate dehydratase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 3-dehydroquinate dehydratase protein or its homologue as shown here, it is used for producing citromalic acid and R-Glyceric acid and/or comprising citromalic acid and the composition of R-Glyceric acid in biological or its part as described, particularly increases citromalic acid and R-Glyceric acid, preferably free or the citromalic acid of combining form and the quantity of R-Glyceric acid.In one embodiment, 3-dehydroquinate dehydratase activity of proteins is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 3-dehydroquinate dehydratase protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b0970 (accession number NP_415490) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be glutamate receptor.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria ybhL protein superfamily, preferably having a purposes of the active protein of glutamate receptor protein matter or its homologue, it is used for producing fine chemicals in biological or its part as described, be trihydroxy-butyric acid and/or the composition that comprises trihydroxy-butyric acid, particularly increase trihydroxy-butyric acid, the preferred quantity of the trihydroxy-butyric acid of free or combining form.In one embodiment, the activity of glutamate receptor is enhanced or produces in the methods of the invention, and for example the activity from colibacillary glutamate receptor or its homologue is enhanced or produces.

The sequence of e. coli k12 b3169 (accession number NP_417638) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Transcription Termination-antitermination factor.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria transcription factor nusA superfamily, preferably having a purposes of the active protein of Transcription Termination-antitermination factor or its homologue, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid or the composition that comprises oxysuccinic acid, particularly increase oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, the activity of Transcription Termination-antitermination factor is enhanced or produces in the methods of the invention, and for example the activity from colibacillary Transcription Termination-antitermination factor or its homologue is enhanced or produces.

The sequence of e. coli k12 b3169 (accession number NP_417638) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Transcription Termination-antitermination factor.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria transcription factor nusA superfamily, preferably having a purposes of the active protein of Transcription Termination-antitermination factor or its homologue, it is used for producing fine chemicals in biological or its part as described, be trihydroxy-butyric acid or the composition that comprises trihydroxy-butyric acid, particularly increase trihydroxy-butyric acid, the preferred quantity of the trihydroxy-butyric acid of free or combining form.In one embodiment, the activity of Transcription Termination-antitermination factor is enhanced or produces in the methods of the invention, and for example the activity from colibacillary Transcription Termination-antitermination factor or its homologue is enhanced or produces.

The sequence of e. coli k12 b3169 (accession number NP_417638) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Transcription Termination-antitermination factor.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of intestinal bacteria transcription factor nusA superfamily, preferably having a purposes of the active protein of Transcription Termination-antitermination factor or its homologue, it is used for producing oxysuccinic acid and trihydroxy-butyric acid or comprising oxysuccinic acid and the composition of trihydroxy-butyric acid in biological or its part as described, particularly increases oxysuccinic acid and trihydroxy-butyric acid, preferably free or the oxysuccinic acid of combining form and the quantity of trihydroxy-butyric acid.In one embodiment, the activity of Transcription Termination-antitermination factor is enhanced or produces in the methods of the invention, and for example the activity from colibacillary Transcription Termination-antitermination factor or its homologue is enhanced or produces.

The sequence of e. coli k12 b3116 (accession number NP_417586) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the L-Threonine/L-Serine permease (HAAAP family) of anaerobic induction.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having Threonine-active gene product of Serine permease superfamily, preferably having L-Threonine/L-Serine permease (HAAAP family) the active protein of anaerobic induction or a purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be succsinic acid and/or the composition that comprises succsinic acid, particularly increase succsinic acid, the preferred quantity of the succsinic acid of free or combining form.In one embodiment, the activity of the L-of anaerobic induction Threonine/L-Serine permease (HAAAP family) is enhanced or produces in the methods of the invention, for example is enhanced or produces from the L-Threonine/L-Serine permease (HAAAP family) of colibacillary anaerobic induction or the activity of its homologue.

The sequence of e. coli k12 b3116 (accession number NP_417586) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the L-Threonine/L-Serine permease (HAAAP family) of anaerobic induction.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having Threonine-active gene product of Serine permease superfamily, preferably having L-Threonine/L-Serine permease (HAAAP family) the active protein of anaerobic induction or a purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and/or the composition that comprises fumaric acid, particularly increase fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, the activity of the L-of anaerobic induction Threonine/L-Serine permease (HAAAP family) is enhanced or produces in the methods of the invention, for example is enhanced or produces from the L-Threonine/L-Serine permease (HAAAP family) of colibacillary anaerobic induction or the activity of its homologue.

The sequence of e. coli k12 b3116 (accession number NP_417586) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the L-Threonine/L-Serine permease (HAAAP family) of anaerobic induction.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having Threonine-active gene product of Serine permease superfamily, preferably having L-Threonine/L-Serine permease (HAAAP family) the active protein of anaerobic induction or a purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid and/or the composition that comprises oxysuccinic acid, particularly increase oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, the activity of the L-of anaerobic induction Threonine/L-Serine permease (HAAAP family) is enhanced or produces in the methods of the invention, for example is enhanced or produces from the L-Threonine/L-Serine permease (HAAAP family) of colibacillary anaerobic induction or the activity of its homologue.

The sequence of e. coli k12 b3116 (accession number NP_417586) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the L-Threonine/L-Serine permease (HAAAP family) of anaerobic induction.Therefore, in one embodiment, the inventive method comprises as shown here having Threonine-active gene product of Serine permease superfamily from colibacillary, preferably have L-Threonine/L-Serine permease (HAAAP family) the active protein of anaerobic induction or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, it is oxysuccinic acid, fumaric acid and/or succsinic acid and/or comprise oxysuccinic acid, the composition of fumaric acid and/or succsinic acid particularly increases oxysuccinic acid, fumaric acid and/or succsinic acid, the oxysuccinic acid of preferred free or combining form, the quantity of fumaric acid and/or succsinic acid.In one embodiment, the activity of the L-of anaerobic induction Threonine/L-Serine permease (HAAAP family) is enhanced or produces in the methods of the invention, for example is enhanced or produces from the L-Threonine/L-Serine permease (HAAAP family) of colibacillary anaerobic induction or the activity of its homologue.

The sequence of e. coli k12 b0057 (accession number NP_414599) has been published in Blattner etc., and Science 277 (5331), 1453-1474, and 1997, and its activity does not characterize yet.Therefore, in one embodiment, the inventive method comprises as shown here from the gene product of the colibacillary b0057 of having protein active or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be R-Glyceric acid, particularly increase R-Glyceric acid or comprise the composition of R-Glyceric acid, the preferred quantity of the R-Glyceric acid of free or combining form.

The sequence of e. coli k12 b4129 (accession number NP_418553) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Methionin tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary Methionin tRNA synthetase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, i.e. succsinic acid and/or comprise the composition of succsinic acid, the preferred quantity of the succsinic acid of free or combining form.In one embodiment, the activity of Methionin tRNA synthetase protein is enhanced or produces in the methods of the invention, and for example the activity from colibacillary Methionin tRNA synthetase protein or its homologue is enhanced or produces.In one embodiment, the activity of Methionin tRNA synthetic enzyme is enhanced or produces in the methods of the invention, and for example the activity from colibacillary Methionin tRNA synthetic enzyme or its homologue is enhanced or produces.

The sequence of e. coli k12 b3129 (accession number NP_417598) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be " the proteolytic enzyme of supposition; HtrA repressor ".Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary " proteolytic enzyme of supposition; The htrA repressor " or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, i.e. and R-Glyceric acid and/or comprise the composition of R-Glyceric acid particularly increases R-Glyceric acid, the preferred quantity of the R-Glyceric acid of free or combining form." the proteolytic enzyme of supposition in the methods of the invention in one embodiment; The htrA repressor " activity be enhanced or produce, for example from the colibacillary " proteolytic enzyme of supposition; The htrA repressor " or the activity of its homologue be enhanced or produce.

The sequence of e. coli k12 b3938 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the biosynthetic transcription repressor of methionine(Met).Therefore, in one embodiment, the inventive method comprises the purposes from the colibacillary biosynthetic transcription repressor of metJ protein superfamily protein, particularly methionine(Met) or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be R-Glyceric acid and/or the composition that comprises R-Glyceric acid, particularly increase R-Glyceric acid, the preferred quantity of the R-Glyceric acid of free or combining form.In one embodiment, the activity of the biosynthetic transcription repressor of methionine(Met) is enhanced or produces in the methods of the invention, and for example the activity from the biosynthetic transcription repressor of colibacillary methionine(Met) or its homologue is enhanced or produces.

The sequence of e. coli k12 b2478 has been published in Blattner, and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the dihydrodipicolinate synthase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary dihydrodipicolinate synthase or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid and/or the composition that comprises oxysuccinic acid, particularly increase the quantity of the oxysuccinic acid of free or combining form.In one embodiment, dihydrodipicolinate synthase's superfamily in the methods of the invention, for example participate in diaminopimelate pathway, the Methionin biosynthesizing, the protein of C-compound and carbohydrate utilization and/or Entner-Doudoroff approach, preferred dihydrodipicolinate synthase's activity is enhanced or produces, for example, for example participate in diaminopimelate pathway from colibacillary dihydrodipicolinate synthase's superfamily, the Methionin biosynthesizing, the protein of C-compound and carbohydrate utilization and/or Entner-Doudoroff approach, the activity of preferred dihydrodipicolinate synthase or its homologue is enhanced or produces.Therefore, in one embodiment, the activity of dihydrodipicolinate synthase or its homologue is enhanced and is used to produce fine chemicals in the methods of the invention, i.e. oxysuccinic acid particularly increases the quantity of the oxysuccinic acid of free or combining form.

The sequence of e. coli k12 b3172 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be argininosuccinate synthetase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary gene product with argininosuccinic acid synthase superfamily protein active, the preferred amino acid bio that participates in synthesizes, nitrogen and sulfo-are thanked, L-glutamic acid family (proline(Pro), oxyproline, arginine, glutamine, L-glutamic acid) biosynthesizing, L-glutamic acid family amino acid degradation, nitrogen and sulphur utilization, ornithine cycle, polyamines and sarkosine biosynthesizing, the aspartate family biosynthesizing, the biosynthetic protein of ammonia assimilation effect and/or L-glutamic acid family, the purposes that more preferably has active proteinic or its homologue of argininosuccinate synthetase, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and/or the composition that comprises fumaric acid, particularly increase fumaric acid, the quantity of the fumaric acid of preferred free or combining form.In one embodiment, the activity of argininosuccinate synthetase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary argininosuccinate synthetase or its homologue is enhanced or produces.

The sequence of e. coli k12 b3172 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be argininosuccinate synthetase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary gene product with argininosuccinic acid synthase superfamily protein active, the preferred amino acid bio that participates in synthesizes, nitrogen and sulfo-are thanked, L-glutamic acid family (proline(Pro), oxyproline, arginine, glutamine, L-glutamic acid) biosynthesizing, L-glutamic acid family amino acid degradation, nitrogen and sulphur utilization, ornithine cycle, polyamines and sarkosine biosynthesizing, the aspartate family biosynthesizing, the biosynthetic protein of ammonia assimilation effect and/or L-glutamic acid family, the purposes that more preferably has active proteinic or its homologue of argininosuccinate synthetase, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid and/or the composition that comprises oxysuccinic acid, particularly increase oxysuccinic acid, the quantity of the oxysuccinic acid of preferred free or combining form.In one embodiment, the activity of argininosuccinate synthetase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary argininosuccinate synthetase or its homologue is enhanced or produces.

The sequence of e. coli k12 b3172 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be argininosuccinate synthetase.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary gene product with argininosuccinic acid synthase superfamily protein active, the preferred amino acid bio that participates in synthesizes, nitrogen and sulfo-are thanked, L-glutamic acid family (proline(Pro), oxyproline, arginine, glutamine, L-glutamic acid) biosynthesizing, L-glutamic acid family amino acid degradation, nitrogen and sulphur utilization, ornithine cycle, polyamines and sarkosine biosynthesizing, the aspartate family biosynthesizing, the biosynthetic protein of ammonia assimilation effect and/or L-glutamic acid family, the purposes that more preferably has active proteinic or its homologue of argininosuccinate synthetase, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid and fumaric acid and/or comprise oxysuccinic acid and the composition of fumaric acid, particularly increase oxysuccinic acid and fumaric acid, preferred free or the oxysuccinic acid of combining form and the quantity of fumaric acid.In one embodiment, the activity of argininosuccinate synthetase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary argininosuccinate synthetase or its homologue is enhanced or produces.

The sequence of e. coli k12 b1961 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be DNA cytosine methylation enzyme.Therefore, in one embodiment, the inventive method comprises as shown here having " site-specific methyltransferase (cytosine specific) EcoRII " active gene product of superfamily from colibacillary, the preferred nucleotide metabolism that participates in, cyclic nucleotide and extraordinary nucleotide metabolism, transcribe, DNA repairs, cytodifferentiation, grow (phylogeny) and/or nucleic acid bonded protein, the purposes that more preferably has protein or its homologue of cytosine methylation enzymic activity, it is used for producing fine chemicals in biological or its part as described, be succsinic acid and/or the composition that comprises succsinic acid, particularly increase succsinic acid, the quantity of the succsinic acid of preferred free or combining form.In one embodiment, the activity of cytosine methylation enzyme is enhanced or produces in the methods of the invention, and for example the activity from colibacillary cytosine methylation enzyme or its homologue is enhanced or produces.

The sequence of e. coli k12 b1961 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be DNA cytosine methylation enzyme.Therefore, in one embodiment, the inventive method comprises as shown here having " site-specific methyltransferase (cytosine specific) EcoRII " active gene product of superfamily from colibacillary, the preferred nucleotide metabolism that participates in, cyclic nucleotide and extraordinary nucleotide metabolism, transcribe, DNA repairs, cytodifferentiation, grow (phylogeny) and/or nucleic acid bonded protein, the purposes that more preferably has protein or its homologue of cytosine methylation enzymic activity, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and/or the composition that comprises fumaric acid, particularly increase fumaric acid, the quantity of the fumaric acid of preferred free or combining form.In one embodiment, the activity of cytosine methylation enzyme is enhanced or produces in the methods of the invention, and for example the activity from colibacillary cytosine methylation enzyme or its homologue is enhanced or produces.

The sequence of e. coli k12 b1961 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be DNA cytosine methylation enzyme.Therefore, in one embodiment, the inventive method comprises as shown here having " site-specific methyltransferase (cytosine specific) EcoRII " active gene product of superfamily from colibacillary, the preferred nucleotide metabolism that participates in, cyclic nucleotide and extraordinary nucleotide metabolism, transcribe, DNA repairs, cytodifferentiation, grow (phylogeny) and/or nucleic acid bonded protein, the purposes that more preferably has protein or its homologue of cytosine methylation enzymic activity, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and succsinic acid and/or comprise fumaric acid and the composition of succsinic acid, particularly increase fumaric acid and succsinic acid, preferred free or the fumaric acid of combining form and the quantity of succsinic acid.In one embodiment, the activity of cytosine methylation enzyme is enhanced or produces in the methods of the invention, and for example the activity from colibacillary cytosine methylation enzyme or its homologue is enhanced or produces.

The sequence of e. coli k12 b2599 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be bifunctional enzyme: chorismate mutase (N-end) and prephenate dehydratase (C-end).Therefore, in one embodiment, the inventive method comprises as shown here having " pheA bifunctional enzyme; prephenate dehydratase homology " active gene product of superfamily from colibacillary, preferred participation halfcystine-aromatic series biosynthesizing, amino acid bio is synthetic, halfcystine-die aromatischen Aminosaeuren degraded and/or VITAMIN, the biosynthetic protein of cofactor and prothetic group, more preferably has bifunctional enzyme: the purposes of chorismate mutase (N-end) and prephenate dehydratase (C-end) active protein or its homologue, it is used for producing fine chemicals in biological or its part as described, be succsinic acid and/or the composition that comprises succsinic acid, particularly increase succsinic acid, the quantity of the succsinic acid of preferred free or combining form.In one embodiment, bifunctional enzyme in the methods of the invention: the activity of chorismate mutase (N-end) and prephenate dehydratase (C-end) is enhanced or produces, and for example from colibacillary bifunctional enzyme: chorismate mutase (N-end) and prephenate dehydratase (C-end) or the activity of its homologue are enhanced or produce.

The sequence of e. coli k12 b4122 has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and it is FURAMIC ACID B (I class hydratase) that its activity is defined as. therefore, in one embodiment, the inventive method comprises as shown here having " iron dependency hydratase; iron dependency tartrate dehydratase α chain homology " active gene product of superfamily from colibacillary, preferably have the protein of FURAMIC ACID B (I class hydratase) or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and/or the composition that comprises fumaric acid, particularly increase fumaric acid, the quantity of the fumaric acid of preferred free or combining form.In one embodiment, the activity of FURAMIC ACID B (I class hydratase) is enhanced or produces in the methods of the invention, for example is enhanced or produces from the colibacillary FURAMIC ACID B (I class hydratase) or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YCL038C has been published in Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and its activity to be defined as be the autophagy associated protein.Therefore, in one embodiment, the inventive method comprises participation from yeast saccharomyces cerevisiae as shown here, and other is transported the gene product of easy beggar (transport facilitator), preferably has a purposes of the active protein of autophagy associated protein or its homologue, it is used for producing fine chemicals in biological or its part as described, be succsinic acid and/or the composition that comprises succsinic acid, particularly increase succsinic acid, the preferred quantity of the succsinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of autophagy associated protein is enhanced or produces, and for example is enhanced or produces from the autophagy associated protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YCL038C has been published in Goffeau, A, Science 274 (5287), 546-547 (1996), Oliver, S.G etc., Nature 357 (6373), 38-46 (1992), and its activity to be defined as be the autophagy associated protein.Therefore, in one embodiment, the inventive method comprises participation from yeast saccharomyces cerevisiae as shown here, and other is transported easy beggar's gene product, preferably has a purposes of the active protein of autophagy associated protein or its homologue, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid and/or the composition that comprises oxysuccinic acid, particularly increase oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, the activity of autophagy associated protein is enhanced or produces in the methods of the invention, for example is enhanced or produces from the autophagy associated protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YCL038C has been published in Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and it is the autophagy associated protein that its activity is defined as. therefore, in one embodiment, the inventive method comprises participation from yeast saccharomyces cerevisiae as shown here, and other transports easy beggar's gene product, the purposes that preferably has the active protein of autophagy associated protein or its homologue, it is used for producing fine chemicals in biological or its part as described, be succsinic acid and oxysuccinic acid and/or comprise succsinic acid and the composition of oxysuccinic acid, particularly increase succsinic acid and oxysuccinic acid, preferred free or the succsinic acid of combining form and the quantity of oxysuccinic acid.In one embodiment, in the methods of the invention, the activity of autophagy associated protein is enhanced or produces, and for example is enhanced or produces from the autophagy associated protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YCR012W has been published in Goffeau, A., and Science 274 (5287), 546-547 (1996), and Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and its activity to be defined as be the glycerol 3-phosphate acid kinase.Therefore, in one embodiment, the inventive method comprises the phosphoglyceric kinase from yeast saccharomyces cerevisiae as shown here-superfamily gene product, preferred participation C-compound and carbohydrate utilization, glycolysis-and glyconeogenesis, sugar, glucoside, the gene product of polyvalent alcohol and carboxylicesters katabolism and/or energy, the purposes that more preferably has protein or its homologue of 3-phoshoglyceric acid kinase activity, it is used for producing fine chemicals in biological or its part as described, be fumaric acid and/or the composition that comprises fumaric acid, particularly increase fumaric acid, the quantity of the fumaric acid of preferred free or combining form.In one embodiment, the kinase whose activity of 3-phoshoglyceric acid is enhanced or produces in the methods of the invention, for example is enhanced or produces from the glycerol 3-phosphate acid kinase of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YOR168W has been published in Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and Goffeau, A, Science 274 (5287), 546-547 (1996), and its activity to be defined as be Glutamine-tRNA ligase.Therefore, in one embodiment, the inventive method comprises the gene product of " human glutamine-tRNA ligase enzyme; Glutamine-tRNA ligase homology " superfamily from yeast saccharomyces cerevisiae as shown here, preferred aminoacyl-tRNA-the synthetic enzyme that participates in, the Nucleotide combination, the protein of translation and/or protein synthesis, the purposes that more preferably has the active protein of Glutamine-tRNA ligase or its homologue, it is used for producing fine chemicals in biological or its part as described, be oxysuccinic acid and/or the composition that comprises oxysuccinic acid, particularly increase oxysuccinic acid, the quantity of the oxysuccinic acid of preferred free or combining form.In one embodiment, the activity of Glutamine-tRNA ligase is enhanced or produces in the methods of the invention, for example is enhanced or produces from the Glutamine-tRNA ligase of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b1896 has been published in Blattner, F.R. etc., and Science 277 (5331), 1453-1474 (1997), and it is defined as having the active protein of trehalose-6-phosphate synthase.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b1896 of e. coli k12 or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, particularly increases citromalic acid, fumaric acid, oxysuccinic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid, the preferred quantity of citromalic acid, fumaric acid, oxysuccinic acid, succsinic acid, three hydroxybutyric acids and/or the trihydroxy-butyric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of trehalose-6-phosphate synthase b1896 is enhanced.

The sequence of yeast saccharomyces cerevisiae YBL015W has been published in Goffeau, A. etc., and Science 274 (5287), 546-547 (1996), and it is defined as having the protein of acetyl-CoA hydrolytic enzyme activities.Therefore, in one embodiment, the inventive method comprises the described active proteinic purposes that has as shown here, it is used for producing each fine chemicals in biological or its part, particularly increases fumaric acid and/or oxysuccinic acid, preferably free or the fumaric acid of combining form and/or the quantity of oxysuccinic acid.In one embodiment, in the methods of the invention, the activity of described acetyl-CoA lytic enzyme YBL015W is enhanced.

The sequence of yeast saccharomyces cerevisiae YCR059C has been published in Goffeau, and Science such as A. 274 (5287), 546-547 (1996), and its activity to be defined as be unclassified protein.Therefore, in one embodiment, the inventive method comprises as shown here from the described unclassified protein of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of non-categorical protein YCR059C is enhanced.

The sequence of yeast saccharomyces cerevisiae YFR007W has been published in Goffeau, and Science such as A. 274 (5287), 546-547 (1996), and its activity to be defined as be unclassified protein.Therefore, in one embodiment, the inventive method comprises as shown here from the described unclassified protein of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of unclassified protein YFR007W is enhanced.

The sequence of YJL055W yeast saccharomyces cerevisiae has been published in Goffeau, and Science such as A. 274 (5287), 546-547 (1996), and its activity to be defined as be lysine decarboxylase sample protein.Therefore, in one embodiment, the inventive method comprises as shown here from the described protein YJL055W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YJL055W is enhanced.

The sequence of yeast saccharomyces cerevisiae YJL099W has been published in Goffeau, and Science such as A. 274 (5287), 546-547 (1996), and its activity to be defined as be to participate in the biosynthetic protein of chitin.Therefore, in one embodiment, the inventive method comprises as shown here from the biosynthetic described protein of participation chitin of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid and/or oxysuccinic acid, preferably free or the fumaric acid of combining form and/or the quantity of oxysuccinic acid.In one embodiment, in the methods of the invention, the activity of protein YJL099W is enhanced.

The sequence of yeast saccharomyces cerevisiae YMR241W has been published in Bowman, S.Nature 387 (6632 supplementary issue), 90-93 (1997), and its activity to be defined as be to have the active Mitochondrial DNA replication protein of yeast suppressor gene (abf2).Therefore, in one embodiment, the inventive method comprises the purposes with the active protein of yeast suppressor gene or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of described protein YMR241W is enhanced.

The sequence of yeast saccharomyces cerevisiae YPR024W has been published in Bussey, Nature such as H. 387 (6632 supplementary issue), 103-105 (1997), and its activity to be defined as be to have the active protein of Mitochondrial ATPase.Therefore, in one embodiment, the inventive method comprises as shown here from the ATP enzyme of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YPR024W is enhanced.

The sequence of yeast saccharomyces cerevisiae YPR138C has been published in Bussey, Nature such as H. 387 (6632 supplementary issue), 103-105 (1997), and its activity to be defined as be to have the active protein of ammonia transporter.Therefore, in one embodiment, the inventive method comprises the purposes with the active protein of ammonia transporter or its homologue from yeast saccharomyces cerevisiae as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid and/or oxysuccinic acid, preferably free or the fumaric acid of combining form and/or the quantity of oxysuccinic acid.In one embodiment, in the methods of the invention, the activity of protein YPR138C is enhanced.

The sequence of e. coli k12 b0730 has been published in Biochem.J.260:737-747 (1989) such as Buck D., and its activity to be defined as be to have the protein that acyl is replied regulatory gene and/or " succinic thiokinase operon transcriptional " function.Therefore, in one embodiment, the inventive method comprises that the acyl that has from e. coli k12 as shown here replys the protein b0730 of regulatory gene function or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, oxysuccinic acid and/or succsinic acid, the preferred quantity of fumaric acid, oxysuccinic acid and/or the succsinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b0730 is enhanced.

The sequence of e. coli k12 b1611 has been published in Blattner, and Science such as F.R. 277 (5331), 1453-1474 (1997), and its activity to be defined as be to have FURAMIC ACID C or the active protein of fumaric acid I class hydratase I.Therefore, in one embodiment, the inventive method comprises the purposes with FURAMIC ACID C or the fumaric acid I class active protein of hydratase I or its homologue from e. coli k12 as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b1611 is enhanced.

The sequence of e. coli k12 b2699 has been published in Blattner, and Science such as F.R. 277 (5331), 1453-1474 (1997), and its activity to be defined as be to have DNA chain exchange and reorganization (RecA) active protein.Therefore, in one embodiment, the inventive method comprises the purposes with the active protein b2699 of RecA or its homologue from e. coli k12 as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, three hydroxybutyric acids and/or trihydroxy-butyric acid, the preferred quantity of fumaric acid, three hydroxybutyric acids and/or the trihydroxy-butyric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b2699 is enhanced.

The sequence of e. coli k12 b4139 has been published in Blattner, and Science such as F.R. 277 (5331), 1453-1474 (1997), and its activity to be defined as be to have the active protein of aspartic acid desaminase (L-Aspartase).Therefore, in one embodiment, the inventive method comprises the purposes with the active protein of aspartic acid desaminase (L-Aspartase) or its homologue from e. coli k12 as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b4139 is enhanced.

The sequence of e. coli k12 b1676 has been published in Blattner, and Science such as F.R. 277 (5331), 1453-1474 (1997), and its activity to be defined as be to have pyruvate kinase I (in the past for F) protein active and that stimulated by fructose.Therefore, in one embodiment, the inventive method comprises the purposes with pyruvate kinase I (being F in the past) active protein or its homologue from e. coli k12 as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid, the preferred quantity of the fumaric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b1676 is enhanced.

The sequence of e. coli k12 b4063 has been published in Blattner, F.R. wait Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be to have soxS redox sensibility activator function and for the protein of the transcriptional activation agent function of peroxide reactions.Therefore, in one embodiment, the inventive method comprise as shown here from e. coli k12 have soxS redox sensibility activator function and for the protein of the transcriptional activation agent function of peroxide reactions or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases fumaric acid and/or oxysuccinic acid, preferably free or the fumaric acid of combining form and/or the quantity of oxysuccinic acid.In one embodiment, in the methods of the invention, the activity of protein b4063 is enhanced.

The sequence of yeast saccharomyces cerevisiae YBR084W has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be the protein with plastosome C1-tetrahydrofolic acid (THFA) synthase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein with plastosome C1-tetrahydrofolic acid (THFA) synthase activity of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YBR084W is enhanced.

The sequence of yeast saccharomyces cerevisiae YBR184W has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be unclassified protein.Therefore, in one embodiment, the inventive method comprises as shown here from the unclassified protein YBR184W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of unclassified protein YBR184W is enhanced.

The sequence of yeast saccharomyces cerevisiae YCL032W has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be the protein that participates in the pheromone signal transduction pathway.Therefore, in one embodiment, the inventive method comprises the protein YCL032W of participation pheromone signal transduction pathway from yeast saccharomyces cerevisiae as shown here or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases oxysuccinic acid and/or succsinic acid, preferably free or the oxysuccinic acid of combining form and/or the quantity of succsinic acid.In one embodiment, in the methods of the invention, the activity of protein YCL032W is enhanced.

The sequence of yeast saccharomyces cerevisiae YGR007W has been published in Tettelin; H. etc.; Nature 387 (6632 supplementary issue); 81-84 (1997), and its activity to be defined as be to have the active protein of phosphocholine two cytidine acyltransferases (being also referred to as phosphorylethanolamine two cytidine acyltransferases or phosphorylcholine two cytidine acyltransferases).Therefore; in one embodiment; the inventive method comprises as shown here from the protein with phosphocholine two cytidine acyltransferase activities of yeast saccharomyces cerevisiae or the purposes of its homologue; it is used for producing each fine chemicals in biological or its part as described, particularly increases oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YGR007W is enhanced.

The sequence of yeast saccharomyces cerevisiae YJL072C has been published in Goffeau, and A.Science 274 (5287), 546-547 (1996), and its function is defined as chromosomal DNA and duplicates required GINS complex subunit protein.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YJL072C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YJL072C is enhanced.

The sequence of yeast saccharomyces cerevisiae YKL132C has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be to have the active protein of the many NADPH-linked glutamate synthase of leaf acyl.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YKL132C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases oxysuccinic acid, the preferred quantity of the oxysuccinic acid of free or combining form.In one embodiment, in the methods of the invention, the YKL132C activity of proteins is enhanced.

The sequence of e. coli k12 b0695 has been published in Blattner, F.R. etc., Science277 (5331), 1453-1474 (1997), and its activity to be defined as be to have the active protein of histidine kinase.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b0695 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases pyruvic acid, the preferred quantity of the pyruvic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b0695 is enhanced.

The sequence of yeast saccharomyces cerevisiae YOR044W has been published in Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and its activity to be defined as be unclassified protein.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YOR044W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases succsinic acid, the preferred quantity of the succsinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YOR044W is enhanced.

The homologous compound (=homologue) of [0023.0.16.16] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with at least a Table I the 3rd row, 190-226 is capable or 564-594 capable shown in protein, for example have by contain Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the protein of the coded peptide sequence of nucleotide sequence of sequence have identical biological function or activity.

In one embodiment, shown in Table II the 3rd row, the 190th or 564 row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given each fine chemicals in the biology, preferred citromalic acid content increase.

In one embodiment, Table II the 3rd row, 191-205 is capable or 565-571 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given each fine chemicals in the biology, preferred fumaric acid content increase.

In one embodiment, Table II the 3rd row, 206-217 is capable or 577-583 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given malic acid content increase in the biology.

In one embodiment, Table II the 3rd row, 218-219 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given three hydroxybutyric acid lactone contents increase in the biology.

In one embodiment, shown in Table II the 3rd row, the 220th row or the 584th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given pyruvic acid content increase in the biology.

In one embodiment, Table II the 3rd row, 221-224 is capable or 585-591 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given succsinic acid content increase in the biology.

In one embodiment, Table II the 3rd row, 225-226 is capable or 592-594 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given trihydroxy-butyric acid content increase in the biology.

In one embodiment, Table II the 3rd row, 572-576 capable shown in the polypeptide homologue of any one be to have same or similar active homologue.Particularly active increasing given R-Glyceric acid content increase in the biology.

[0023.1.0.16] as Table II the 3rd row, 190-226 is capable or the homologue of the polypeptide of 564-594 shown in capable can be by Table I the 7th row, 190-226 is capable or 564-594 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, 190-226 is capable or 564-594 capable shown in polypeptide.

The homologue of the polypeptide shown in Table II the 3rd row, the 190th or 564 row can be have increase citromalic acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, the 190th or 564 row or Table II the 7th row, the 190th or 564 row.

As Table II the 3rd row, 191-205 is capable or the homologue of the polypeptide of 565-571 shown in capable can be have increase fumaric acid content and/or quantity is active, by Table I the 7th row, 191-205 is capable or 565-571 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, 191-205 is capable or 565-571 capable shown in polypeptide.

As Table II the 3rd row, 206-217 is capable or the homologue of the polypeptide of 577-583 shown in capable can be have increase malic acid content and/or quantity is active, by Table I the 7th row, 206-217 is capable or 577-583 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, 206-217 is capable or 577-583 capable shown in polypeptide.

The homologue of the polypeptide shown in Table II the 3rd row, the 218th and 219 row can be have increase by three hydroxybutyric acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, the 218th and 219 row or Table II the 7th row, the 218th and 219 row.

The homologue of the polypeptide shown in Table II the 3rd row, the 220th row or the 584th row can be have increase pyruvic acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, the 220th row or the 584th row or Table II the 7th row, the 220th row or the 584th row.

As Table II the 3rd row, 221-224 is capable or the homologue of the polypeptide of 585-591 shown in capable can be have increase succsinic acid content and/or quantity is active, by Table I the 7th row, 221-224 is capable or 585-591 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, 221-224 is capable or 585-591 capable shown in polypeptide.

As the homologue of the polypeptide shown in Table II the 3rd row, the 225th and 226 row or 592-594 are capable can be have increase trihydroxy-butyric acid content and/or quantity is active, by Table I the 7th row, the 225th and 226 row or 592-594 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, the 225th and 226 row or 592-594 capable shown in polypeptide.

[0024.0.0.16] sees [0024.0.0.0]

[0025.0.16.16] is according to the present invention, R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid level increase in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II the 3rd row, 190-226 is capable or 564-594 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, 190-226 is capable or 564-594 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, 190-226 is capable or 564-594 capable shown in any one proteinic biology or the enzyme activity, if promptly with Table II the 3rd row, 190-226 is capable or 564-594 capable shown in any one protein compare, its have at least initial enzymic activity 10%, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if polypeptide come comfortable evolve go up with biophase shown in Table I the 4th row apart from nearer biological and in evolution with in the biology of initial biological apart from each other, expressing, polypeptide of the present invention can be given described activity, as increase each fine chemicals in biological or its part.For example just eozoan is biological from different sections, order, guiding principle or door with expression, and biology comes from identical section, order, guiding principle or door shown in first eozoan and Table I the 4th row.

[0025.1.0.16] and [0025.2.0.16] sees [0025.1.0.0] and [0025.2.0.0]

[0025.3.16.16] in one embodiment, if polypeptide come comfortable evolve go up with biophase shown in Table I the 4th row apart from nearer biological and in evolution with in the biology of initial biological apart from each other, expressing, the used polypeptide of polypeptide of the present invention or the inventive method can be given described activity, as increase each fine chemicals in biological or its part.For example just eozoan is biological from different sections, order, guiding principle or door with expression, and biology comes from identical section, order, guiding principle or door shown in first eozoan and Table I the 4th row.

[0026.0.0.16] to [0033.0.0.16]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.16.16] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or by Table I the 5th row, 190-226 is capable or 564-594 capable shown in the protein of nucleic acid molecule encoding or its homologue (as Table I the 7th row, 190-226 is capable or 564-594 capable shown in homologue) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows each fine chemicals quantity that increases.

[0035.0.0.16] to [0044.0.0.16]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.16.16] in one embodiment, at e. coli k12 protein b0057 or its homologue (shown in Table II the 5th or 7 row, the 572nd row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferably glycerine acid be increased in 35% and 57% or more between.

In one embodiment, at e. coli k12 protein b0161 or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 565th row of fumaric acid or for the 577th of oxysuccinic acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferred fumaric acid and/or oxysuccinic acid increases, preferably give fumaric acid be increased in 64% and 151% between and/or oxysuccinic acid be increased in 64% and 229% or more between.

In one embodiment, at e. coli k12 protein b0970 or its homologue (shown in Table II the 5th or 7 row, the 592nd row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred trihydroxy-butyric acid be increased in 34% and 80% or more between.

In one embodiment, at e. coli k12 protein b1343 or its homologue (shown in Table II the 5th or 7 row, the 593rd row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred trihydroxy-butyric acid be increased in 33% and 108% or more between.

In one embodiment, at e. coli k12 protein b1693 or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 564th row of citromalic acid or for the 573rd of R-Glyceric acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferably glycerine acid and/or citromalic acid increases, preferably give R-Glyceric acid be increased in 33% and 52% between and/or citromalic acid be increased in 48% and 105% or more between.

In one embodiment, at e. coli k12 protein b1738 or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 566th row of fumaric acid or for the 585th of succsinic acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferred succsinic acid and/or fumaric acid increases, preferably give succsinic acid be increased in 49% and 95% between and/or fumaric acid be increased in 53% and 149% or more between.

In one embodiment, at e. coli k12 protein b1961 or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 567th row of fumaric acid or for the 586th of succsinic acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferred succsinic acid and/or fumaric acid increases, preferably give succsinic acid be increased in 29% and 54% between and/or fumaric acid be increased in 76% and 105% or more between.

In one embodiment, at e. coli k12 protein b2478 or its homologue (shown in Table II the 5th or 7 row, the 578th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferably apple acid be increased in 51% and 124% or more between.

In one embodiment, at e. coli k12 protein b2599 or its homologue (shown in Table II the 5th or 7 row, the 587th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred succsinic acid be increased in 33% and 156% or more between.

In one embodiment, (be listed as at e. coli k12 protein b3116 or its homologue as Table II the 5th or 7, for the 588th row of succsinic acid or for the 568th row of fumaric acid or for shown in the 579th row of oxysuccinic acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred succsinic acid and/or fumaric acid and/or oxysuccinic acid increase, preferably give succsinic acid be increased in 31% and 42% between and/or fumaric acid be increased in 63% and 99% between and/or oxysuccinic acid be increased in 60% and 212% or more between.

In one embodiment, at e. coli k12 protein b3129 or its homologue (shown in Table II the 5th or 7 row, the 574th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferably glycerine acid be increased in 30% and 35% or more between.

In one embodiment, at e. coli k12 protein b3160 or its homologue (shown in Table II the 5th or 7 row, the 589th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred succsinic acid be increased in 33% and 90% or more between.

In one embodiment, at e. coli k12 protein b3169 or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 580th row of oxysuccinic acid or for the 594th of trihydroxy-butyric acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferably apple acid and/or trihydroxy-butyric acid increases, preferably give oxysuccinic acid be increased in 132% and 143% between and/or trihydroxy-butyric acid be increased in 60% and 88% or more between.

In one embodiment, at e. coli k12 protein b3172 or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 581st row of oxysuccinic acid or for the 569th of fumaric acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferred fumaric acid and/or oxysuccinic acid increases, preferably give fumaric acid be increased in 63% and 325% between and/or oxysuccinic acid be increased in 64% and 408% or more between.

In one embodiment, at e. coli k12 protein b3231 or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 575th row of R-Glyceric acid or for the 584th of pyruvic acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferred pyruvic acid and/or R-Glyceric acid increases, preferably give pyruvic acid be increased in 43% and 46% between and/or R-Glyceric acid be increased in 21% and 34% or more between.

In one embodiment, at e. coli k12 protein b3938 or its homologue (shown in Table II the 5th or 7 row, the 576th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferably glycerine acid be increased in 37% and 40% or more between.

In one embodiment, at e. coli k12 protein b4122 or its homologue (shown in Table II the 5th or 7 row, the 570th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred fumaric acid be increased in 108% and 430% or more between.

In one embodiment, at e. coli k12 protein b4129 or its homologue (shown in Table II the 5th or 7 row, the 590th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred succsinic acid be increased in 40% and 84% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YCL038C or its homologue (shown in Table II the 5th or 7 is listed as, goes for the 591st row of succsinic acid or for the 582nd of oxysuccinic acid), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably giving fine chemicals, preferred succsinic acid and/or oxysuccinic acid increases, preferably give succsinic acid be increased in 47% and 91% between and/or oxysuccinic acid be increased in 87% and 123% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YCR012W or its homologue (shown in Table II the 5th or 7 row, the 571st row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferred fumaric acid be increased in 62% and 93% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR168W or its homologue (shown in Table II the 5th or 7 row, the 583rd row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably give fine chemicals, preferably apple acid be increased in 56% and 562% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b1896 or its homologue (shown in Table II the 5th or 7 row, the 190th row) is enhanced, give each fine chemicals, preferred citromalic acid be increased in 63% and 99% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YBL015W or its homologue (shown in Table II the 5th or 7 row, the 191st row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 50% and 126% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YCR059C or its homologue, for example shown in Table II the 5th or 7 row, the 192nd row under the activity of proteins situation about being enhanced, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 71% and 183% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YFR007W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 193rd row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 69% and 156% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL055W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 194th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 72% and 561% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL099W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 195th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 98% and 734% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YMR241W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 196th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 75% and 368% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPR024W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 197th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 97% and 374% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPR138C or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 198th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 61% and 327% or more between.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 199th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 75% and 838% or more between.

In one embodiment, at e. coli k12 protein b1611 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 200th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 106% and 328% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 201st row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 65% and 802% or more between.

In one embodiment, at e. coli k12 protein b4139 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 202nd row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 118% and 525% or more between.

In one embodiment, at e. coli k12 protein b1676 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 203rd row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 108% and 430% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 204th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 232% and 320% or more between.

In one embodiment, at e. coli k12 protein b4063 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 205th row, preferably, give in one embodiment each fine chemicals, preferred fumaric acid be increased in 64% and 308% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBL015W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 206th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 45% and 86% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR084W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 207th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 107% and 448% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR184W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 208th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 67% and 213% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YCL032W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 209th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 87% and 123% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YGR007W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 210th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 55% and 90% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL072C or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 211st row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 49% and 249% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL099W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 212nd row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 94% and 797% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YKL132C or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 213rd row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 109% and 213% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPR138C or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 214th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 72% and 359% or more between.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 215th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 96% and 672% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 216th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 149% and 319% or more between.

In one embodiment, at e. coli k12 protein b4063 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 217th row, preferably, give in one embodiment each fine chemicals, preferably apple acid be increased in 78% and 93% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 218th row, preferably, give in one embodiment each fine chemicals, preferred three hydroxybutyric acids be increased in 48% and 215% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 219th row, preferably, give in one embodiment each fine chemicals, preferred three hydroxybutyric acids be increased in 34% and 96% or more between.

In one embodiment, at e. coli k12 protein b0695 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 220th row, preferably, give in one embodiment each fine chemicals, preferred pyruvic acid be increased in 61% and 155% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YCL032W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 221st row, preferably, give in one embodiment each fine chemicals, preferred succsinic acid be increased in 47% and 91% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR044W or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 222nd row, preferably, give in one embodiment each fine chemicals, preferred succsinic acid be increased in 45% and 265% or more between.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 223rd row, preferably, give in one embodiment each fine chemicals, preferred succsinic acid be increased in 51% and 332% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 224th row, preferably, give in one embodiment each fine chemicals, preferred succsinic acid be increased in 55% and 110% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 225th row, preferably, give in one embodiment each fine chemicals, preferred trihydroxy-butyric acid be increased in 82% and 281% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that activity of proteins is enhanced shown in Table II the 5th or 7 row, the 226th row, preferably, give in one embodiment each fine chemicals, preferred trihydroxy-butyric acid be increased in 60% and 161% or more between.

[0046.0.16.16] is as disclosed protein in [0016.0.16.16] or its homologue (shown in Table I the 5th or 7 row, the 190th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred citromalic acid and other organic acid increases.

As disclosed protein in [0016.0.16.16] or its homologue (shown in Table I the 5th or 7 row, 191-205 are capable), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred fumaric acid and other organic acid increases.

As disclosed protein in [0016.0.16.16] or its homologue (shown in Table I the 5th or 7 row, 206-217 are capable), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferably apple acid and other organic acid increases.

As disclosed protein in [0016.0.16.16] or its homologue (shown in Table I the 5th or 7 row, the 218th or 219 row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred three hydroxybutyric acid lactones and other organic acid increases.

As disclosed protein in [0016.0.16.16] or its homologue (shown in Table I the 5th or 7 row, the 220th row), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred pyruvic acid and other organic acid increases.

As disclosed protein in [0016.0.16.16] or its homologue (shown in Table I the 5th or 7 row, 221-224 are capable), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred succsinic acid and other organic acid increases.

As disclosed protein in [0016.0.16.16] or its homologue (shown in Table I the 5th or 7 row, 225-226 are capable), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred trihydroxy-butyric acid and other organic acid increases.

As disclosed e. coli k12 protein b0057 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 572nd the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferably glycerine acid and other organic acid increases.

As disclosed e. coli k12 protein b0161 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for fumaric acid the 565th the row and/or for oxysuccinic acid the 577th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred fumaric acid and/or oxysuccinic acid and other organic acid increases.

As disclosed e. coli k12 protein b0970 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 592nd the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred trihydroxy-butyric acid and other organic acid increases.

As disclosed e. coli k12 protein b1343 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 593rd the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred trihydroxy-butyric acid and other organic acid increases.

As disclosed e. coli k12 protein b1693 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for R-Glyceric acid the 573rd the row and/or for citromalic acid the 564th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferably glycerine acid and/or citromalic acid and other organic acid increase.

As disclosed e. coli k12 protein b1738 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for fumaric acid the 566th the row and/or for succsinic acid the 585th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred fumaric acid and/or succsinic acid and other organic acid increases.

As disclosed e. coli k12 protein b1961 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for fumaric acid the 567th the row and/or for succsinic acid the 586th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred fumaric acid and/or succsinic acid and other organic acid increases.

As disclosed e. coli k12 protein b2478 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 578th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferably apple acid and other organic acid increases.

As disclosed e. coli k12 protein b2599 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 587th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred succsinic acid and other organic acid increases.

As disclosed e. coli k12 protein b3116 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for succsinic acid the 588th the row and/or for fumaric acid the 568th the row and/or for oxysuccinic acid the 579th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred succsinic acid, fumaric acid and/or oxysuccinic acid and other organic acid increases.

As disclosed e. coli k12 protein b3129 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 574th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferably glycerine acid and other organic acid increases.

As disclosed e. coli k12 protein b3160 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 589th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred succsinic acid and other organic acid increases.

As disclosed e. coli k12 protein b3169 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for trihydroxy-butyric acid the 594th the row and/or for oxysuccinic acid the 580th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred trihydroxy-butyric acid and/or oxysuccinic acid and other organic acid increases.

As disclosed e. coli k12 protein b3172 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for fumaric acid the 569th the row and/or for oxysuccinic acid the 581st the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred fumaric acid and/or oxysuccinic acid and other organic acid increases.

As disclosed e. coli k12 protein b3231 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for R-Glyceric acid the 575th the row and/or for pyruvic acid the 584th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, give each fine chemicals, preferably glycerine acid and/or pyruvic acid and other organic acid increase.

As disclosed e. coli k12 protein b3938 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 576th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferably glycerine acid and other organic acid increases.

As disclosed e. coli k12 protein b4122 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 570th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred fumaric acid and other organic acid increases.

As disclosed e. coli k12 protein b4129 or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 590th the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred succsinic acid and other organic acid increases.

As disclosed yeast saccharomyces cerevisiae protein YCL038C or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, for succsinic acid the 591st the row and/or for oxysuccinic acid the 582nd the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred succsinic acid and/or oxysuccinic acid and other organic acid increases.

As disclosed yeast saccharomyces cerevisiae protein YCR012W or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 571st the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferred fumaric acid and other organic acid increases.

As disclosed yeast saccharomyces cerevisiae protein YOR168W or its homologue in [0016.0.16.16] (as Table I the 5th or 7 row, the 583rd the row shown in), for example have such as in [0022.0.16.16] under the active activity of proteins of the definition situation about being enhanced, preferably, giving each fine chemicals, preferably apple acid and other organic acid increases.

[0047.0.0.16] to [0048.0.0.16]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.16.16] has to give and improves each fine chemicals citromalic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in the 190th row or the 564th row and/or as Table II the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in the 190th row or the 564th row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 190th row or the 564th row) sequence of coded polypeptide, and have the activity that the citromalic acid level of giving described in the literary composition increases.

Have to give and improve fumaric acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in the capable or 565-571 of 191-205 is capable and/or as Table II the 5th or 7 row, its function homologue described in polypeptide or the literary composition shown in the capable or 565-571 of 191-205 is capable, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the capable or 565-571 of 191-205 is capable) sequence of coded polypeptide, and have the activity that the fumaric acid level of giving described in the literary composition increases.

Have to give and improve R-Glyceric acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as the sequence of consensus sequence shown in Table IV the 7th row, 572-576 are capable and/or its function homologue described in polypeptide shown in Table II the 5th or 7 row, 572-576 are capable or literary composition or by the sequence of the coded polypeptide of nucleic acid molecule that this paper characterized or nucleic acid molecule of the present invention (for example by Table I the 5th or 7 row, 572-576 capable shown in nucleic acid molecule or its function homologue as herein described), and have the activity that the R-Glyceric acid level of giving described in the literary composition increases.

Have to give and improve oxysuccinic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in the capable or 577-583 of 206-217 is capable and/or as Table II the 5th or 7 row, its function homologue shown in 577-583 is capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the capable or 577-583 of 206-217 is capable) sequence of coded polypeptide, and have the activity that the oxysuccinic acid level of giving described in the literary composition increases.

Have to give and improve each fine chemicals three hydroxybutyric acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the sequence of consensus sequence shown in Table IV the 7th row, the 218th or 219 row and/or shown in Table II the 5th or 7 row, the 218th or 219 row its function homologue described in polypeptide or the literary composition or by the sequence of the coded polypeptide of nucleic acid molecule that this paper characterized or nucleic acid molecule of the present invention (for example by nucleic acid molecule or its function homologue as herein described shown in Table I the 5th or 7 row, the 218th or 219 row), and have the activity that the three hydroxybutyric acid levels of giving described in the literary composition increase.

Have to give and improve each fine chemicals pyruvic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in the 220th row or the 584th row and/or as Table II the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in the 220th row or the 584th row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 220th row or the 584th row) sequence of coded polypeptide, and have the activity that the pyruvic acid level of giving described in the literary composition increases.

Have to give and improve each fine chemicals succsinic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in the capable or 585-591 of 221-224 is capable and/or as Table II the 5th or 7 row, its function homologue described in polypeptide or the literary composition shown in the capable or 585-591 of 221-224 is capable, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the capable or 585-591 of 221-224 is capable) sequence of coded polypeptide, and have the activity that the succsinic acid level of giving described in the literary composition increases.

Have to give and improve each fine chemicals trihydroxy-butyric acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in the capable or 592-594 of 225-226 is capable and/or as Table II the 5th or 7 row, its function homologue described in polypeptide or the literary composition shown in the capable or 592-594 of 225-226 is capable, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the capable or 592-594 of 225-226 is capable) sequence of coded polypeptide, and have the activity that the trihydroxy-butyric acid level of giving described in the literary composition increases.

[0050.0.16.16] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, the sylvite of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid or sodium salt or their ester or glucoside (for example its diglucoside).

[0051.0.16.16] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise the composition of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of organic acid or its salt (for example R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid).

[0052.0.0.16] sees [0052.0.0.0]

[0053.0.16.16] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein give nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded protein matter or polypeptide of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide) express and increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide) express and improve;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide) express and improve;

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide);

(h) by add positive Expression element or remove negative Expression element improve code book invention polypeptide or the used polypeptide of the inventive method (for example have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near gene of the present invention or the used gene of the inventive method and makes gene of the present invention or used genetic expression of the inventive method so enhanced strain system;

(i) to strengthen code book invention protein or the expression of used proteinic gene of the inventive method or protein itself or the growth conditions that active mode is regulated and control plant, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein of the present invention or the used protein active of the inventive method and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.16.16] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improve coded protein or have as Table II the 3rd or 5 row, 190-226 is capable or 564-594 capable shown in protein or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide) give each fine chemicals after active expression of polypeptides or the activity and increase.

[0055.0.0.16] to [0067.0.0.16]: see that [0055.0.0.0] is to [0067.0.0.0]

[0068.0.16.16] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid.

[0069.0.0.16] sees [0069.0.0.0]

[0070.0.16.16] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used expression of polypeptides of the inventive method, perhaps a gene of code book invention protein or the used polypeptide of the inventive method or a plurality of gene import biology separately or with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example contain high level (from the physiology of nutrition angle) R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, the favourable citromalic acid of three hydroxybutyric acids and/or trihydroxy-butyric acid or their derivative, R-Glyceric acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid or its biological chemistry derivative composition.

[0071.0.0.16] sees [0071.0.0.0]

[0072.0.16.16]

[0073.0.16.16] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant; With

(b) improve polypeptide of the present invention or the used polypeptide of the inventive method or its homologue (for example Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves; With

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation.

[0074.0.16.16] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded or free and each fine chemicals of bonded.

[0075.0.0.16] to [0077.0.0.16]: see that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.16.16] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes.Can pass through method known to those skilled in the art purified fermentation broth, tunning, plant or plant product.

For these different methods of setting up gradually, product is R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid or the composition that contains R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.16] to [0084.0.0.16]: see that [0079.0.0.0] is to [0084.0.0.0]

[0084.0.16.16]-/-

[0085.0.16.16] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.16] to [0087.0.0.16]: see that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.16.16] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Stronger and output is enhanced to the resistance of biological and abiotic stress because the nutritive value that for example is used for the plant of raise poultry depends on above-mentioned fine chemicals or plant, so this is very important for the plant breeder.。

[0088.1.0.16] sees [0088.1.0.0]

[0089.0.0.16] to [0094.0.0.16]: see that [0089.0.0.0] is to [0094.0.0.0]

[0095.0.16.16] advantageously increases R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid pond to separate a large amount of pure basically fine chemicals and/or to obtain the resistance of the raising of biology and abiotic stress and obtain high yield by method of the present invention in genetically modified organism.

[0096.0.16.16] in another embodiment preferred of the present invention, raising the present invention or the used nucleotide sequence of the inventive method or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals in the biology) are combined and be can be used for producing each fine chemicals.

[0097.0.16.16]-/-

[0098.0.16.16] in preferred embodiments, each fine chemicals be produce according to the present invention and carry out isolating where necessary.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.10.16].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition that comprises each fine chemicals.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.16.16] comprises the transgenic plant that the method according to this invention synthetic comprises fine chemicals such as R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid and can advantageously directly put on market, and do not need institute's synthetic fine chemicals is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet each fine chemicals that the method according to this invention produces can also be as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free fine chemicals from biology, advantageously separate from plant.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid are positioned at cell in the microorganism, must relate to separation to their recovery to biomass.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.

[0101.0.10.16] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 11 27-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.16.16] for example, R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid can advantageously detect by HPLC, LC or GC separation method.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect the existence of the product that contains R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

[0103.0.16.16] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as Table II the 5th or 7 row, 190-226 is capable or the sequence of 564-594 shown in capable;

Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I the 5th or 7 row, 190-226 is capable or the sequence of 564-594 shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use have as Table III the 7th row, 190-226 is capable or 564-594 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, 190-226 is capable or 564-594 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.18.16] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule IIA the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide of sequence.

[00103.2.0.16] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule IIB the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide of sequence.

[0104.0.16.16] in one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide of sequence.

[0105.0.0.16] to [0107.0.0.16]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.16.16] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in the capable or 564-594 of 190-226 is capable, from Table II the 5th or 7 row, aminoacid sequence shown in the capable or 564-594 of 190-226 is capable is derived or from containing Table IV the 7th row, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in the capable or 564-594 of 190-226 is capable, perhaps its coding has as Table II the 3rd, 5 or 7 row, the enzymic activity of polypeptide or bioactive polypeptide shown in the capable or 564-594 of 190-226 is capable or for example to give each fine chemicals behind its expression or active the increasing be R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, derivative or homologue that three hydroxybutyric acids and/or trihydroxy-butyric acid increase.

[0109.0.16.16] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make each fine chemicals, particularly R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid that the inventive method produced be optimized synthetic.

[0110.0.0.16] sees [0110.0.0.0]

[0111.0.0.16] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.16.16] the inventive method is the isolated nucleic acid sequences form, its coding have as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in polypeptide active polypeptide or have as Table II the 5th and 7 row, 190-226 is capable or 564-594 capable shown in the polypeptide of peptide sequence, and give R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid level and increase.

[0113.0.0.16] to [0114.0.0.16]: see that [0113.0.0.0] is to [0114.0.0.0]

[0115.0.0.16] sees [0115.0.0.0]

[0116.0.0.16] to [0120.0.0.16] sees that [0116.0.0.0] is to [0120.0.0.0]

[0120.1.0.16]：-/-

[0121.0.16.16] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, peptide sequence shown in 190-226 is capable or its functional homologue as herein described have the difference of one or more amino acid moleculars, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, giving the citromalic acid level after the activity of peptide sequence shown in the 190th row or the 564th row increases, perhaps improving Table II the 5th or 7 row, giving the fumaric acid level shown in the capable or 565-571 of 191-205 is capable after the activity of peptide sequence increases, perhaps improving Table II the 5th or 7 row, giving the R-Glyceric acid level shown in 572-576 is capable after the activity of peptide sequence increases, perhaps improving Table II the 5th or 7 row, giving the oxysuccinic acid level shown in the capable or 577-583 of 206-217 is capable after the activity of peptide sequence increases, perhaps improving Table II the 5th or 7 row, give the increase of three hydroxybutyric acid levels shown in the 218th or 219 row after the activity of peptide sequence, perhaps improving Table II the 5th or 7 row, giving the pyruvic acid level after the activity of peptide sequence shown in the 220th or 584 row increases, perhaps improving Table II the 5th or 7 row, giving the succsinic acid level shown in the capable or 585-591 of 221-224 is capable after the activity of peptide sequence increases, and is perhaps improving Table II the 5th or 7 row, giving the trihydroxy-butyric acid level after the activity of peptide sequence shown in the 225th and 226 row or 592-594 are capable increases.

[0122.0.0.16] to [0127.0.0.16]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.16.16] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 190-226 is capable or the primer of 564-594 shown in capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence or from as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence deutero-sequence produce.

[0129.0.16.16] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (sequence of polypeptide particularly of the present invention or the used polypeptide of the inventive method).The conservative region of polypeptide of the present invention or the used polypeptide of the inventive method is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, 190-226 is capable or 564-594 capable shown in consensus sequence from described comparison.

[0130.0.16.16] can use degenerated primer to have the new protein fragment of aforementioned activity (giving R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid increase as comprise this segmental protein expression or activity in raising after) by pcr amplification then.

[0131.0.0.16] to [0138.0.0.16]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.16.16] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with for the Table I the 5th of citromalic acid or 7 row, the 190th row or the 564th row or for the Table I the 5th of fumaric acid or 7 row, 191-205 is capable or 565-571 is capable or for the Table I the 5th of oxysuccinic acid or 7 row, 206-217 is capable or 577-583 is capable or for the Table I the 5th of trihydroxy-butyric acid or 7 row, the 218th or 219 row or for the Table I the 5th of pyruvic acid or 7 row, the 220th row or the 584th row or for the Table I the 5th of succsinic acid or 7 row, 221-224 is capable or 585-591 is capable or for the Table I the 5th of trihydroxy-butyric acid or 7 row, the 225th or 226 or 592-594 capable shown in sequence hybridization, and the coding expression has each fine chemicals, the i.e. R-Glyceric acid of increasing, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, the active peptide of three hydroxybutyric acids and/or trihydroxy-butyric acid.

[0140.0.0.16] to [0146.0.0.16]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.16.16] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise one of the nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.16.16] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 5th or 7 row, nucleotide sequence or its portion homologous were at least about 30% shown in the capable or 564-594 of 190-226 was capable, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly having after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue increases R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, the activity of three hydroxybutyric acids and/or trihydroxy-butyric acid.

[0149.0.16.16] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, one of nucleotide sequence shown in the capable or 564-594 of 190-226 is capable or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and (as gives R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid increase) and randomly as Table II the 5th row, 190-226 is capable or 564-594 is capable, preferred Table II B, the 7th row, the protein of protein active shown in the capable or 564-594 of 190-226 is capable.

[00149.1.0.16] randomly, in one embodiment, with Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, 190-226 is capable or 564-594 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, 190-226 is capable or the protein of 564-594 shown in capable is known activity or is used for these protein of note.

[0150.0.16.16] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, the part of the coding region of one of nucleotide sequence shown in the capable or 564-594 of 190-226 is capable, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in one of sequence sense strand, Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.

Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 190-226 is capable or 564-594 capable shown in the right PCR of primer will produce as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th is listed as, 190-226 is capable or 564-594 capable shown in fragment or its gene product of polynucleotide sequence.

[0151.0.0.16]: see [0151.0.0.0]

[0152.0.16.16] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coded polypeptide or its part, described polypeptide or its part comprise and are listed as Table II the 5th or 7, the abundant homology of aminoacid sequence shown in the capable or 564-594 of 190-226 is capable, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising citromalic acid as be shown in the examples (the 190th row) or fumaric acid (191-205 or 565-571 are capable) or oxysuccinic acid (the capable or 577-583 of 206-217) or trihydroxy-butyric acid (the 218th or 219 row) or pyruvic acid (the 220th row or the 584th row) or succsinic acid (221-224 is capable or 585-591 is capable) or trihydroxy-butyric acid (the 225th or 226 row) level.

[0153.0.16.16] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in protein or its part have Table II the 3rd row for example as herein described, 190-226 is capable or 564-594 capable shown in the activity of polypeptide.

[0154.0.16.16] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.16] to [0156.0.0.16]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.16.16] the present invention relate in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase), for example comprise as Table IV the 5th or 7 row, 190-226 is capable or 564-594 capable shown in consensus sequence polypeptide or as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment as Table IV the 5th or 7 row, 190-226 is capable or the consensus sequence of 564-594 shown in capable or as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 5th or 7 row, 190-226 is capable or the consensus sequence of 564-594 shown in capable or as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I A the 7th row, 190-226 is capable or the sequence of 564-594 shown in capable.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in nucleic acid molecule identical.

[0158.0.0.16] to [0160.0.0.16]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.16.16] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.16] sees [0162.0.0.0]

[0163.0.16.16] preferably, under stringent condition with Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the nucleic acid molecule of the present invention of sequence hybridization or the inventive method nucleic acid molecule used therefor corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals quantity increase in biology or its part (for example tissue, cell or cell chamber) after using activity of proteins).

[0164.0.0.16] sees [0164.0.0.0]

[0165.0.16.16] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.16] to [0167.0.0.16]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.16.16] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, 190-226 is capable or 564-594 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, 190-226 is capable or the aminoacid sequence of 564-594 shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, sequence shown in the capable or 564-594 of 190-226 is capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, one of sequence shown in the capable or 564-594 of 190-226 is capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, sequence shown in the capable or 564-594 of 190-226 is capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, sequence shown in the capable or 564-594 of 190-226 is capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.16] to [0172.0.0.16]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.16.16] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:17198 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:17198 sequence before use.

[0174.0.0.16]: see [0174.0.0.0]

[0175.0.16.16] for example, the sequence that has 80% homology at protein level and SEQ ID NO:17199 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:17199 sequence.

[0176.0.16.16] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in the capable or 564-594 of 190-226 was capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in the capable or 564-594 of 190-226 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in the capable or 564-594 of 190-226 is capable has essentially identical character and discerns.

[0177.0.16.16] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, the functional equivalent that nucleotide sequence obtained shown in the capable or 564-594 of 190-226 was capable be listed as according to of the present invention as Table II the 5th or 7,190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, one of polypeptide had at least 30% shown in the capable or 564-594 of 190-226 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, the polypeptide of the essentially identical character of polypeptide shown in the capable or 564-594 of 190-226 is capable.

[0178.0.0.16] sees [0178.0.0.0]

[0179.0.16.16] can by to the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in) in introduce replacement, interpolation or the disappearance of one or more Nucleotide, and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th is listed as, 190-226 is capable or 564-594 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to encoding sequence, for example as Table I the 5th or 7 row, 190-226 is capable or 564-594 introduces sudden change in the sequence of nucleotide sequence shown in capable.

[0180.0.0.16] to [0183.0.0.16]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.16.16] is employed to have as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, the nucleotide sequence homologous compound of sequence shown in the capable or 564-594 of 190-226 is capable, perhaps come Table II the 5th or 7 row freely, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, the homologous compound of the nucleotide sequence of sequence shown in the capable or 564-594 of 190-226 is capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.16.16] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, 190-226 is capable or the sequence of 564-594 shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, 190-226 is capable or the arbitrary sequence of 564-594 shown in capable in other Nucleotide of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, 190-226 is capable or the sequence of 564-594 shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.16.16] also preferred the inventive method comprise as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, 190-226 is capable or 564-594 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, 190-226 is capable or 564-594 capable shown in sequence identical.

[0187.0.16.16] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, 190-226 is capable or 564-594 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, 190-226 is capable or 564-594 capable shown in the encoding sequence of sequence identical.

[0188.0.16.16] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, 190-226 is capable or 564-594 is capable, preferred Table II the 5th or 7 row, shown in the capable or 564-594 of 190-226 is capable and under the same conditions the polypeptide expressed activity is compared and is not reduced substantially.In one embodiment, polypeptide of the present invention be comprise as Table I B the 7th row, 190-226 is capable or 564-594 capable shown in sequence or by its homologue of forming.

[0189.0.16.16] as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the homologous compound of sequence, or deutero-as Table II the 5th or 7 row, 190-226 is capable or the homologue of the sequence of 564-594 shown in capable also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.16]: see [0190.0.0.0]

[0191.0.0.16] sees [0191.0.0.0]

Biological or its part of [0191.0.16.16] and described contrast or selection is compared, should biology or its part produces free and/or each fine chemicals level of bonded improves according to the inventive method described herein.

[0192.0.0.16] to [0203.0.0.16]: see that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.16.16] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding is being listed as Table II the 5th or 7 of mature form at least, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, polypeptide or its segmental nucleic acid molecule shown in the capable or 564-594 of 190-226 is capable, described nucleic acid molecule is given each fine chemicals in biological or its part, i.e. R-Glyceric acid (572-576 is capable) or acid of lemon lush (the 190th or 564 row) or fumaric acid (the capable or 565-571 of 191-205) or oxysuccinic acid (the capable or 577-583 of 206-217) or trihydroxy-butyric acid (the 218th or 219 row) or pyruvic acid (the 220th row or the 584th row) or succsinic acid (221-224 is capable or 585-591 is capable) or trihydroxy-butyric acid (the 225th or 226 or 592-594 capable) quantity increases;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, 190-226 is capable or nucleic acid molecule or its segmental nucleic acid molecule of 564-594 shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 5th or 7 row, the nucleic acid molecule that primer shown in the capable or 564-594 of 190-226 is capable or primer obtain amplification from cDNA library or genomic library, and in biological or its part, give each fine chemicals, promptly R-Glyceric acid (572-576 is capable) or citromalic acid (the 190th or 564 row) or fumaric acid (the capable or 565-571 of 191-205) or oxysuccinic acid (the capable or 577-583 of 206-217) or trihydroxy-butyric acid (the 218th or 219 row) or pyruvic acid (the 220th row or the 584th row) or succsinic acid (221-224 is capable or 585-591 is capable) or trihydroxy-butyric acid (the 225th or 226 or 592-594 capable) increase of quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like Table IV the 7th row, consensus sequence shown in the capable or 564-594 of 190-226 is capable is also given each fine chemicals in biological or its part, promptly R-Glyceric acid (572-576 is capable) or citromalic acid (the 190th or 564 row) or fumaric acid (the capable or 565-571 of 191-205) or oxysuccinic acid (the capable or 577-583 of 206-217) or trihydroxy-butyric acid (the 218th or 219 row) or pyruvic acid (the 220th row or the 584th row) or succsinic acid (221-224 is capable or 585-591 is capable) or trihydroxy-butyric acid (the 225th or 226 or 592-594 capable) nucleic acid molecule that increases of quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be R-Glyceric acid (572-576 is capable) or citromalic acid (the 190th or 564 row) or fumaric acid (the capable or 565-571 of 191-205) or oxysuccinic acid (the capable or 577-583 of 206-217) or trihydroxy-butyric acid (the 218th or 219 row) or pyruvic acid (the 220th row or the 584th row) or succsinic acid (221-224 is capable or 585-591 is capable) or trihydroxy-butyric acid (the 225th or 226 or 592-594 capable) nucleic acid molecule that increases of quantity, described peptide coding such as Table II the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table II B the 7th row, the structural domain of the polypeptide shown in the capable or 564-594 of 190-226 is capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7,190-226 is capable or 564-594 is capable, preferred Table I B the 7th row, nucleic acid molecule shown in the capable or 564-594 of 190-226 is capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in the capable or 564-594 of 190-226 is capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt; Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby, the nucleic acid molecule of preferred (a) to (l) by one or more Nucleotide be different from as Table I A the 5th or 7 row, 190-226 is capable or the sequence of 564-594 shown in capable.In one embodiment, nucleic acid molecule of the present invention not by Table I A the 5th or 7 row, 190-226 is capable or the sequence of 564-594 shown in capable formed.In another embodiment, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as Table II A the 5th or 7 row, 190-226 is capable or the peptide sequence of 564-594 shown in capable.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A the 5th or 7 row, 190-226 is capable or the polypeptide of 564-594 shown in capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as Table II A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the protein of sequence.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in protein sequence at least 30% identical and with Table II A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

Yet, in another embodiment, (a) to the nucleic acid molecule of (l) by one or more Nucleotide be different from as Table I B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence.In one embodiment, nucleic acid molecule of the present invention not by Table I B the 5th or 7 row, 190-226 is capable or the sequence of 564-594 shown in capable formed.In another embodiment, nucleic acid molecule of the present invention and Table I B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In other embodiments, nucleic acid molecule coding schedule IIB the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II B the 5th or 7 row, 190-226 is capable or the polypeptide of 564-594 shown in capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as Table II B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the protein of sequence.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence form.In another embodiment, protein of the present invention and Table II B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in protein sequence at least 30% identical and with Table II B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.16] to [0206.0.0.16]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.16.16] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of glutamic acid metabolism, phosphoenolpyruvic acid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.16] to [0226.0.0.16]: see that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.16.16] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 190-226 is capable or the capable sequence or derivatives thereof of mentioning of 564-594, can be advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one L-glutamic acid or phosphoenolpyruvic acid pathways metabolism in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.16.16] therefore cultivated to cross simultaneously in another embodiment of the present invention and expressed coding and at least one nucleic acid of L-glutamic acid or the direct or indirect related protein of phosphoenolpyruvic acid metabolism or the biology of a gene.

[0229.0.0.0]％

[0230.0.0.16] sees [230.0.0.0]

[0231.0.16.16] is in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.16] to [0276.0.0.16]: see that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.16.16] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.

[0278.0.0.16] to [0282.0.0.16]: see that [0278.0.0.0] is to [0282.0.0.0]

[0283.0.16.16] in addition, can from cell, separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in protein, perhaps as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the antibody of polypeptide or its antigen part, it can comprise above-mentioned sequence or by its polypeptide of forming by the standard technique utilization, and polypeptide for example of the present invention or its fragment produce.Preferably specificity in conjunction with as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the monoclonal antibody of polypeptide.

[0284.0.0.16] sees [0284.0.0.0]

[0285.0.16.16] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence by as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.16.16] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in the capable or 564-594 of 190-226 is capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in the capable or 564-594 of 190-226 is capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, 190-226 is capable or 564-594 capable shown in the polypeptide of consensus sequence.

[0287.0.0.16] to [0289.0.0.16]: see that [0287.0.0.0] is to [0289.0.0.0]

[00290.0.16.16] uses Vector NTI Suite 8.0, (InforMax ^TM, Invitrogen ^TMLifescience software, U.S.Main Office, 7305 Executive Way, Frederick, MD21704, U.S.) assembly AlignX software (on September 25th, 2002) and use following setting to compare: for the pairing comparison: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Right for multiple ratio: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Breach separates the point penalty scope: 8; Residue is replaced matrix: blosum62; Hydrophilic residue: G P S N D Q E K R; Conversion weight: 0.5; Total calculation options: total residue mark: 0.9.Also selected to set in advance in order to compare conserved amino acid.

[0291.0.16.16] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the polypeptide that the present invention relates to comprise plant or the special consensus sequence of microorganism or especially form.

In one embodiment, described polypeptide of the present invention by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 190-226 is capable or the sequence of 564-594 shown in capable.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in the capable or 564-594 of 190-226 is capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably 20% or 10% hydrogen base acid at the most and be different from as Table II A or IIB the 5th or 7 row, sequence shown in the capable or 564-594 of 190-226 is capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence form.

[0292.0.0.16] sees [0292.0.0.0]

[0293.0.16.16] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 190-226 is capable or 564-594 capable shown in other sequence of sequence phase region.In another embodiment, polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence form.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide not by Table I A or IB the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.16.16] in one embodiment, the present invention relates to have as Table II the 3rd row, the polypeptide of activity of proteins shown in the capable or 564-594 of 190-226 is capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in the capable or 564-594 of 190-226 is capable.

[0295.0.0.16] to [0296.0.0.16]: see that [0295.0.0.0] is to [0296.0.0.0]

[0297.0.0.16] sees [0297.0.0.0]

The chemical of [00297.1.0.16] non-polypeptide of the present invention is for example not have Table II the 3rd, 5 or 7 row, the activity of polypeptide and/or the polypeptide of aminoacid sequence shown in the capable or 564-594 of 190-226 is capable.

[0298.0.16.16] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the identical aminoacid sequence of sequence.

[0299.0.16.16] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in one of nucleotide sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.16.16] is therefore, and be described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,190-226 is capable or 564-594 capable shown in sequence different.Therefore, this polypeptide contain with as Table II A or B the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.16] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.16.16] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence shown in the capable or 564-594 of 190-226 is capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.16] sees [0303.0.0.0]

[0304.0.16.16] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause generation have basically as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.16] to [0308.0.0.16]: see that [0305.0.0.0] is to [0308.0.0.0]

[00306.1.0.0] in one embodiment, compound is for comprising each fine chemicals, the fine chemicals of promptly described organic acid or recovery, this organic acid composition of promptly free or protein bound form.

[0309.0.16.16] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, protein shown in the capable or 564-594 of 190-226 is capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " other polypeptide " during the capable or 564-594 of 190-226 is capable is meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, polypeptide shown in the capable or 564-594 of 190-226 is capable is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, it is proteinic and shown in the capable or 564-594 of 190-226 is capable from the protein of identical or different biology.In one embodiment, be not shown in " other polypeptide " that Table II the 5th or 7 is listed as, 190-226 is capable or 564-594 is capable and do not give each fine chemicals increase in biology or its part.In one embodiment, be not shown in " the non-polypeptide of the present invention " or " other polypeptide " that Table II the 5th or 7 is listed as, 190-226 is capable or 564-594 is capable and do not give each fine chemicals increase in biology or its part.

[0310.0.0.16] to [0334.0.0.16] but[0318.0.18.18]: see[0310.0.0.0] to [0334.0.0.0]

[0318.0.18.18] in an especially preferred embodiment, polypeptide of the present invention does not have those proteinic sequences capable as Table II 190-226 or that the capable listed sequence of 564-594 is coded in addition.

[0335.0.16.16] confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, 190-226 is capable or the expression of the nucleotide sequence of 564-594 shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.16] to [0342.0.0.16]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.16.16] as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.16] to [0361.0.0.16]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.16.16] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, polypeptide shown in the capable or 564-594 of 190-226 is capable, for example coding has protein such as Table II the 3rd row, the polypeptide of polypeptide active shown in the capable or 564-594 of 190-226 is capable) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention or used polypeptide of the inventive method or Nucleotide improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Have as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in polypeptide (for example have as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

[0363.0.0.16] sees [0363.0.0.0]

[0364.0.16.16] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette--for example naturally occurring coding as Table II the 3rd row, 190-226 is capable or 564-594 capable shown in polypeptide of the present invention gene promotor with accordingly as Table I the 5th row, 190-226 is capable or 564-594 capable shown in the combination of protein coding sequence--become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.16] to[0373.0.0.16]: see that [0365.0.0.0] is to [0373.0.0.0]

[0374.0.16.16] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the R-Glyceric acid that produces in the inventive method, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid, particularly each fine chemicals also can be from plant with free glycerol acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or each fine chemicals of trihydroxy-butyric acid, particularly free or be bonded to compound or the isolated in form of part (for example glucoside, for example diglucoside).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.16] to [0376.0.0.16]: see that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.16.16] therefore the invention still further relates to the method according to this invention, separated the R-Glyceric acid that is produced, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid thus.

[0378.0.16.16] can separate by this way and be higher than 50% by weight, favourable is higher than 60%, preferably be higher than 70%, especially preferably be higher than 80%, extremely preferably be higher than 90% the R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or the trihydroxy-butyric acid that produce in the method.As required, resultant R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.16.16] in one embodiment, γ-An Jidingsuan and shikimic acid are the mixtures of each fine chemicals.

The suitable synthetic parent material of the R-Glyceric acid that [0380.0.16.16] obtains in the method for the invention, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid composition that separation produces or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is the R-Glyceric acid that the inventive method produces, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid or genetically modified organism are at animal-feed, food, medicine, foodstuff additive, purposes in makeup or the medicine or be used to produce R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, the purposes of three hydroxybutyric acids and/or trihydroxy-butyric acid, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

[0381.0.0.16] to [0382.0.0.16]: see that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.16.16]-/-

[0384.0.0.16] sees [0384.0.0.0]

The fermented liquid that [0385.0.16.16] obtains with this kind approach, particularly contain fermented liquid with other organic acid, amino acid, polypeptide or polysaccharide blended R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid, usually dry matter content accounts for the 1-70% of weight, preferred 7.5 to 25%.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means and to utilize the concentration of sugar during this period of time to remain on or be reduced to 0-10g/l in the fermention medium, preferred to 0-3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.16.16] therefore can also be further purified the R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or the trihydroxy-butyric acid that produce according to the present invention.For this reason, the composition that will contain product separates (by for example open column chromatography or HPLC), and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.16] to [0392.0.0.16]: see that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.16.16] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 190-226 is capable or 564-594 is capable, preferred Table I B the 5th or 7 row, 190-226 is capable or the nucleic acid molecule of 564-594 shown in capable hybridized under lax stringent condition nucleic acid molecule, and randomly separates full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.16] to [0398.0.0.16]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.16.16] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving each fine chemicals generation raising under these conditions.

[00399.1.0.16] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing whether this effect depends on as Table II the 5th or 7 row, 190-226 is capable or the polypeptide of 564-594 shown in capable or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.16] to [0415.0.0.16]: see that [0400.0.0.0] is to [0415.0.0.0]

[0416.0.0.16] sees [0416.0.0.0]

[0417.0.16.16] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor to R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid biosynthetic pathway has resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid synthetic inhibitor.

[0418.0.0.16] to [0423.0.0.16]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.16.16] therefore, nucleic acid of the present invention or the inventive method nucleic acid molecule used therefor, polypeptide of the present invention or the used polypeptide of the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other organic acids.

Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.16] to [0434.0.0.0]: see that [0425.0.0.0] is to [0434.0.0.0]

[0435.0.16.16] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.16.16] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.16.16] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.16.16] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.16.16] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.16.16] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci USA. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.16] to[0443.0.0.16] see that [0441.0.0.0] is to [0443.0.0.0]

[0444.0.16.16] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.16.16] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.16] to [0450.0.0.16]: see that [0446.0.0.0] is to [0450.0.0.0]

[0451.0.0.16] sees [0451.0.5.5]

[0452.0.0.16] to [0453.0.0.16]: see that [0452.0.0.0] is to [0453.0.0.0]

[0454.0.16.16] embodiment 8: the influence that analyzing nucleic acid molecules produces R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid

[0455.0.16.16] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the purpose product of cellular component (is a R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid) raising that produces measures plant, fungi, genetic modification is to purpose compound (R-Glyceric acid for example in algae or the ciliate, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid) influence that produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

[0456.0.0.16]: see [0456.0.0.0]

[0457.0.16.16] embodiment 9: the purifying of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid

[0458.0.16.16] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS or TLC can clearly detect the existence of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid product to the biology analysis of recombinating.The biological total quantity that yeast produced that for example is used for the inventive method can be according to the following step analysis:

By ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed such as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 402 2S1V diluters with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.17ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, used Hewlett Packard 1100HPLC, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Technologies, can be from Ultra Scientific Inc., 250Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6x250mm[Waters, 34 Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

[0459.0.16.16] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids and/or trihydroxy-butyric acid.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.0.16] sees [0460.0.0.0]

[0461.0.16.16] embodiment 10: clone SEQ ID NO:17198,17818,16100,17898,15872,15848,15780,18092,18380,17104,16824,17374,16284,16410,17712,16132,16128,16118,16014,17920,17948,17198,17038 or 18088 or shown in other gene be used for expressing plant

[0462.0.0.16] sees [0462.0.0.0]

[0463.0.16.16] as described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation by pcr amplification SEQ ID NO:17198,17818,16100,17898,15872,15848,15780,18092,18380,17104,16824,17374,16284,16410,17712,16132,16128,16118,16014,17920,17948,17198,17038 or 18088 or shown in other gene.

[0464.0.0.0.16] to [0466.0.0.16]: see that [0464.0.0.0] is to [0466.0.0.0]

[0466.1.0.16] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.16.16] selects following primer sequence for genes of SEQ ID NO:17198:

I) forward primer (SEQ ID NO:17372)

atgagtcgtt?tagtcgtagt?atcta

Ii) reverse primer (SEQ ID NO:17373)

ttacgcaagc?tttggaaagg?tagc

Select following primer sequence for genes of SEQ ID NO:17818:

I) forward primer (SEQ ID NO:17896)

atgacaattt?ctaatttgtt?aaagcag

Ii) reverse primer (SEQ ID NO:17897)

ctagtcaact?ggttcccagc?tgt

Select following primer sequence for genes of SEQ ID NO:16100:

I) forward primer (SEQ ID NO:16116)

atggatgacg?atcacgaaca?gttg

Ii) reverse primer (SEQ ID NO:16117)

ttacgagtcg?aagccggccc?t

Select following primer sequence for genes of SEQ ID NO:17898:

I) forward primer (SEQ ID NO:17918)

atggtcgata?cgcataaact?agca

Ii) reverse primer (SEQ ID NO:17919)

ctagtcgttg?cggatatgga?cga

Select following primer sequence for genes of SEQ ID NO:15872:

I) forward primer (SEQ ID NO:16012)

atgacaatgg?aaaaaaatgg?aggtaa

Ii) reverse primer (SEQ ID NO:16013)

ttatttagca?caatcctcgt?gagc

Select following primer sequence for genes of SEQ ID NO:15848:

I) forward primer (SEQ ID NO:15870)

atgaatttgt?tttggccatc?ggaaa

Ii) reverse primer (SEQ ID NO:15871)

tcaaggttgt?actgtgtgat?ctag

Select following primer sequence for genes of SEQ ID NO:15780:

I) forward primer (SEQ ID NO:15846)

atgccatcta?ccactaatac?tgct

Ii) reverse primer (SEQ ID NO:15847)

ctaatgtttg?gcaactgggg?tttc

Select following primer sequence for genes of SEQ ID NO:18092:

I) forward primer (SEQ ID NO:18378)

atgaacgttt?caaaaatact?tgtgtc

Ii) reverse primer (SEQ ID NO:18379)

tcatgcattt?aacattgtag?gaatttt

Select following primer sequence for genes of SEQ ID NO:18380:

I) forward primer (SEQ ID NO:18640)

atggctcggg?gtgacggaca?t

Ii) reverse primer (SEQ ID NO:18641)

tcatgcttct?tttgcgtgat?gcaat

Select following primer sequence for genes of SEQ ID NO:17104:

I) forward primer (SEQ ID NO:17196)

atgggacaca?agcccttata?ccg

Ii) reverse primer (SEQ ID NO:17197)

ttatcgcgat?gattttcgct?gcg

Select following primer sequence for genes of SEQ ID NO:16824:

I) forward primer (SEQ ID NO:17036)

atgaatacag?tacgcagcga?aaa

Ii) reverse primer (SEQ ID NO:17037)

ttaacgcccg?gctttcatac?tgc

Select following primer sequence for genes of SEQ ID NO:17374:

I) forward primer (SEQ ID NO:17710)

atggctatcg?acgaaaacaa?acag

Ii) reverse primer (SEQ ID NO:17711)

ttaaaaatct?tcgttagttt?ctgctac

Select following primer sequence for genes of SEQ ID NO:16284:

I) forward primer (SEQ ID NO:16408)

atgtgtttaa?agcaaatcat?tggcag

Ii) reverse primer (SEQ ID NO:16409)

ttactgttcg?ctttcatcag?tatag

Select following primer sequence for genes of SEQ ID NO:16410:

I) forward primer (SEQ ID NO:16822)

atgaaaaaga?ccaaaattgt?ttgca

Ii) reverse primer (SEQ ID NO:16823)

ttacaggacg?tgaacagatg?cgg

Select following primer sequence for genes of SEQ ID NO:17712:

I) forward primer (SEQ ID NO:17816)

atggaaaaga?aattaccccg?catta

Ii) reverse primer (SEQ ID NO:17817)

ttagttttgt?tcatcttcca?gcaag

Select following primer sequence for genes of SEQ ID NO:17818:

I) forward primer (SEQ ID NO:17896)

atgacaattt?ctaatttgtt?aaagcag

Ii) reverse primer (SEQ ID NO:17897)

ctagtcaact?ggttcccagc?tgt

Select following primer sequence for genes of SEQ ID NO:16132:

I) forward primer (SEQ ID NO:16282)

atgttgtcga?gactatcttt?attgag

Ii) reverse primer (SEQ ID NO:16283)

ttaaaataga?ccttcaattt?caccgt

Select following primer sequence for genes of SEQ ID NO:16128:

I) forward primer (SEQ ID NO:16130)

atgtaccaaa?ataatgtatt?gaatgct

Ii) reverse primer (SEQ ID NO:16131)

tcaatagtgc?attaactctc?ccatt

Select following primer sequence for genes of SEQ ID NO:16118:

I) forward primer (SEQ ID NO:16126)

atggaggacg?gtaaacaggc?cat

Ii) reverse primer (SEQ ID NO:16127)

ttagagtctt?ccaccggggg?tg

Select following primer sequence for genes of SEQ ID NO:16014:

I) forward primer (SEQ ID NO:16098)

atgacggtaa?acttagatcc?ggata

Ii) reverse primer (SEQ ID NO:16099)

ttatatggac?attccctttt?tttggt

Select following primer sequence for genes of SEQ ID NO:17920:

I) forward primer (SEQ ID NO:17946)

atgtcactac?cggcacattt?gca

Ii) reverse primer (SEQ ID NO:17947)

ttaaatattg?aattcttctt?catcatttt

Select following primer sequence for genes of SEQ ID NO:17948:

I) forward primer (SEQ ID NO:18086)

atggatgata?taagcggaag?gcaaa

Ii) reverse primer (SEQ ID NO:18087)

ctatactggc?aagtgacagt?tgtg

Select following primer sequence for genes of SEQ ID NO:17038:

I) forward primer (SEQ ID NO:17102)

atgaataacg?aacccttacg?tccc

Ii) reverse primer (SEQ ID NO:17103)

ttacatatcc?tcatgaaatt?cttcaagt

Select following primer sequence for genes of SEQ ID NO:16118:

I) forward primer (SEQ ID NO:16126)

atggaggacg?gtaaacaggc?cat

Ii) reverse primer (SEQ ID NO:16127)

ttagagtctt?ccaccggggg?tg

Select following primer sequence for genes of SEQ ID NO:18088:

I) forward primer (SEQ ID NO:18090)

atgatagagg?cgttggaaat?agttc

Ii) reverse primer (SEQ ID NO:18091)

ttatatcgat?cctgcgatat?aataac

The more primers that are used to clone nucleic acid shown in Table II the 5th row see Table III the 7th row.

[0468.0.16.16] to [0470.0.16.16]: see that [0468.0.0.0] is to [0470.0.0.0]

The PCR product that [0470.1.16.16] utilizes the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBIFermentas) phosphorylation to produce by Pfu Turbo archaeal dna polymerase, and the clone enters in the binary vector of treated mistake.

[0471.0.16.16] sees [0471.0.0.0]

[0471.1.16.16] uses Pfu Turbo archaeal dna polymerase benefit flat in the second step building-up reactions by the DNA end of the PCR product that the Herculase archaeal dna polymerase produces.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Utilize the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBI Fermentas) phosphorylation PCR product, and be cloned in the carrier of handling.

[0472.0.16.16] to [0479.0.16.16]: see that [0472.0.0.0] is to [0479.0.0.0]

[0480.0.16.16] embodiment 11: express SEQ ID NO:17198,17818,16100,17898,15872,15848,15780,18092,18380,17104,16824,17374,16284,16410,17712,16132,16128,16118,16014,17920,17948,17198,17038 or 18088 or the generation of the transgenic plant of gene shown in Table I the 5th row

[0481.0.0.16] to [0513.0.0.16]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.16.16] alternatively, as Farr é, E. etc., Plant Physiol, 2001, the 127 the volume, the described detection organic acid of 685-700 page or leaf.

The different plants of being analyzed the results are shown in following table 1:

Table 1

?ORF	Metabolite	Method	??Min	??Max
					?b1896	Citromalic acid	?GC	??1.63	??1.99
?YBL015W	Fumaric acid	?LC	??1.50	??2.26
					?YCR059C	Fumaric acid	?GC	??1.71	??2.83
?YFR007W	Fumaric acid	?GC	??1.69	??2.56
					?YJL055W	Fumaric acid	?GC	??1.72	??6.61
?YJL099W	Fumaric acid	?GC	??1.98	??8.34
					?YMR241W	Fumaric acid	?GC+LC	??1.75	??4.68
?YPR024W	Fumaric acid	?GC	??1.97	??4.74

??YPR138C	Fumaric acid	??LC	??1.61	??4.27
					??b0730	Fumaric acid	??GC+LC	??1.75	??9.38
??b1611	Fumaric acid	??GC+LC	??2.06	??4.28
					??b2699	Fumaric acid	??GC	??1.65	??9.02
??b4139	Fumaric acid	??GC+LC	??2.18	??6.25
					??b1676	Fumaric acid	??GC	??2.08	??5.30
??b1896	Fumaric acid	??GC+LC	??3.32	??4.20
					??b4063	Fumaric acid	??GC	??1.64	??4.08
??YBL015W	Oxysuccinic acid	??LC	??1.45	??1.86
					??YBR084W	Oxysuccinic acid	??GC	??2.07	??5.48
??YBR184W	Oxysuccinic acid	??LC	??1.67	??3.13
					??YCL032W	Oxysuccinic acid	??LC	??1.87	??2.23
??YGR007W	Oxysuccinic acid	??LC	??1.55	??1.90
					??YJL072C	Oxysuccinic acid	??GC	??1.49	??3.49
??YJL099W	Oxysuccinic acid	??GC	??1.94	??8.97
					??YKL132C	Oxysuccinic acid	??GC	??2.09	??3.13
??YPR138C	Oxysuccinic acid	??GC	??1.72	??4.59
					??b0730	Oxysuccinic acid	??GC+LC	??1.96	??7.72
??b1896	Oxysuccinic acid	??GC	??2.49	??4.19
					??b4063	Oxysuccinic acid	??GC	??1.78	??1.93
??b2699	Three hydroxybutyric acids	??GC	??1.48	??3.15
					??b1896	Three hydroxybutyric acids	??GC	??1.34	??1.96
??b0695	Pyruvic acid	??GC	??1.61	??2.55
					??YCL032W	Succsinic acid	??LC	??1.47	??1.91
??YOR044W	Succsinic acid	??LC	??1.45	??3.65
					??b0730	Succsinic acid	??LC	??1.51	??4.32

?b1896	Succsinic acid	??GC+LC	??1.55	??2.10
					?b2699	Trihydroxy-butyric acid	??GC	??1.82	??3.81
?b1896	Trihydroxy-butyric acid	??GC	??1.60	??2.61
					?b1693	Citromalic acid	??GC	??1.48	??2.05
?b0161	Fumaric acid	??LC	??1.64	??2.51
					?b1738	Fumaric acid	??LC	??1.53	??2.49
?b1961	Fumaric acid	??GC+LC	??1.76	??2.05
					?b3116	Fumaric acid	??GC	??1.63	??1.99
?b3172	Fumaric acid	??GC	??1.63	??4.25
					?b4122	Fumaric acid	??GC	??2.08	??5.30
?YCR012W	Fumaric acid	??LC	??1.62	??1.93
					?b0057	R-Glyceric acid	??GC	??1.35	??1.57
?b1693	R-Glyceric acid	??GC	??1.33	??1.52
					?b3129	R-Glyceric acid	??GC	??1.30	??1.35
?b3231	R-Glyceric acid	??GC	??1.21	??1.34
					?b3938	R-Glyceric acid	??GC	??1.37	??1.40
?b0161	Oxysuccinic acid	??GC	??1.64	??3.29
					?b2478	Oxysuccinic acid	??GC+LC	??1.51	??2.24
?b3116	Oxysuccinic acid	??GC	??1.60	??3.12
					?b3169	Oxysuccinic acid	??GC	??2.32	??2.43
?b3172	Oxysuccinic acid	??GC	??1.64	??5.08
					?YCL038C	Oxysuccinic acid	??LC	??1.87	??2.23
?YOR168W	Oxysuccinic acid	??GC+LC	??1.56	??6.62
					?b3231	Pyruvic acid	??GC	??1.43	??1.46
?b1738	Succsinic acid	??LC	??1.49	??1.95
					?b1961	Succsinic acid	??LC	??1.29	??1.54

?b2599	Succsinic acid	??GC+LC	??1.33	??2.56
					?b3116	Succsinic acid	??LC	??1.31	??1.42
?b3160	Succsinic acid	??LC	??1.33	??1.90
					?b4129	Succsinic acid	??LC	??1.40	??1.84
?YCL038C	Succsinic acid	??LC	??1.47	??1.91
					?b0970	Trihydroxy-butyric acid	??GC	??1.34	??1.80
?b1343	Trihydroxy-butyric acid	??GC	??1.33	??2.08
					?b3169	Trihydroxy-butyric acid	??GC	??1.60	??1.88

The 2nd row have shown organic acid citromalic acid, fumaric acid, oxysuccinic acid, trihydroxy-butyric acid, trihydroxy-butyric acid, succsinic acid, the pyruvic acid of being analyzed.The 4th row and the 5th row have shown the ratio of organic acid between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

[0515.0.0.16] to [0552.0.0.16]: see that [0515.0.0.0] to [0552.0.0.0] comprises [0530.1.0.0] to [0530.6.0.0] and [0552.2.0.0]

[0552.1.18.18] embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

??ORF	Metabolite	??Min	??Max
				??b2699	Fumaric acid	??1.38	??1.54
??YJL055W	Fumaric acid	??1.42	??2.01
				??YMR241W	Fumaric acid	??1.75	??4.89
??b1611	Fumaric acid	??1.42	??1.69
				??YBR084W	Oxysuccinic acid	??1.73	??11.39
??b1896	Three hydroxybutyric acid lactones	??1.59	??2.01

Table 2 shows that organic acid has increased in the genetic modification maize plant of expressing e. coli k12 nucleotide sequence b2699, b1611, b1896 or yeast saccharomyces cerevisiae nucleotide sequence YJL055W, YMR241W or YBR084W.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example have under the situation that the activity of the active protein of recombinant protein recA (preferably participating in DNA reorganization and DNA reparation, pheromone reaction, mating type decision, sex-specific protein, Nucleotide combination) is enhanced, preferably, give fumaric acid be increased in 38% and 54% or more between.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that the activity of trehalose-6-phosphate synthase is enhanced, give fine chemicals, preferred trihydroxy-butyric acid be increased in 59% and 101% or more between.

In one embodiment, at e. coli k12 protein b1611 or its homologue, for example fumarate hydratase superfamily protein (preferably participates in the C-compound, sugar metabolism, tricarboxylic acid approach (tricarboxylic acid cycle, the Krebs circulation, the TCA circulation), energy metabolism, the aspartic acid biosynthesizing, the aspartic acid degraded, nitrogen and sulphur utilization, aspartic acid family amino acid degradation, pyruvic acid family (L-Ala, Isoleucine, leucine, Xie Ansuan) and the biosynthesizing of d-L-Ala or nitrogen and sulphur utilization regulate, be preferably FURAMIC ACID C (fumaric acid II class hydratase)) activity situation about being enhanced under, preferably, give fumaric acid be increased in 42% and 69% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL055W or its homologue, for example the protein of the conservative putative protein matter YJL055w superfamily of yeast (preferably participates in the Methionin biosynthesizing, be preferably lysine decarboxylase sample protein) activity situation about being enhanced under, preferably, give fumaric acid be increased in 42% and 101% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YMR241W or its homologue, ADP for example, the protein of ATP carrier protein superfamily (preferably participates in C-compound, carbohydrate transhipment and/or mitochondrial transport, be preferably the Mitochondrial DNA replication protein) activity situation about being enhanced under, preferably, give fumaric acid be increased in 74% and 389% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR084W or its homologue, for example (it is synthetic preferably to participate in amino acid bio for " C1-tetrahydrofolic acid (THFA) synthase; formic acid-tetrahydrofolic acid (THFA) ligase enzyme homology; methylenetetrahydrofolate dehydrogenase (NAD+) homology " superfamily protein, metabolism of purine nucleotide, VITAMIN, cofactor and prothetic group biosynthesizing, C-compound and carbohydrate utilization, tRNA modifies, the Histidine biosynthesizing, the Histidine degraded, the aspartate family biosynthesizing, winter propylhomoserin family amino acid degradation, Nucleotide combination and/or nucleotide metabolism, be preferably C1-tetrahydrofolic acid (THFA) synthase) activity situation about being enhanced under, preferably, give oxysuccinic acid be increased in 73% and 1039% or more between.

[0553.0.16.16]

1. produce the method for R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid, it comprises:

A) improve in non-human being or its one or more parts or produce as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in the activity of protein or its function equivalent; With

B) in allowing described biology, produce under the condition of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid and cultivate this biology.

2. produce the method for R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide or its fragment, described nucleic acid molecule gives R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity increases;

B) contain just like Table I the 5th or 7 row, 190-226 is capable or the nucleic acid molecule of the nucleic acid molecule of 564-594 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 5th or 7 row, 190-226 is capable or 564-594 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity and increase;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 5th or 7 row, 190-226 is capable or 564-594 capable shown in consensus sequence polypeptide and give biology or its part in R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or the increase of trihydroxy-butyric acid quantity, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or the trihydroxy-butyric acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, 190-226 is capable or 564-594 capable shown in polypeptide or its fragment, described nucleic acid molecule gives R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity increases;

B) contain just like Table I the 5th or 7 row, 190-226 is capable or the nucleic acid molecule of the nucleic acid molecule of 564-594 shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 190-226 is capable or 564-594 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity and increase;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 190-226 is capable or 564-594 capable shown in consensus sequence polypeptide and give biology or its part in quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give R-Glyceric acid in biology or its part, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or the increase of trihydroxy-butyric acid quantity, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, 190-226 is capable or 564-594 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 190-226 is capable or 564-594 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the agonist of screening polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or the trihydroxy-butyric acid level that will measure or expression of polypeptides level and standard R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and give the method that R-Glyceric acid in plant or the microorganism, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, the polypeptide that three hydroxybutyric acids or trihydroxy-butyric acid quantity increase and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and it is coded and give R-Glyceric acid in biology or its part to be provided at the nucleic acid molecule of described read-out system of permission and claim 6, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, response compound and described polypeptide bonded detectable signal under the condition of the expression of polypeptides that three hydroxybutyric acids or trihydroxy-butyric acid quantity increase; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and give the method that R-Glyceric acid in the cell, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or the trihydroxy-butyric acid level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give R-Glyceric acid in the host cell, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid level after it is expressed and improve with wild-type.

20. identify and give the method that R-Glyceric acid in the cell, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or the trihydroxy-butyric acid level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give R-Glyceric acid in the host cell, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid level after it is expressed and improve with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid level at biology.

25. agrochemicals, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. any one described method of claim 1-5, the nucleic acid molecule of claim 6, the polypeptide of claim 14, the nucleic acid construct of claim 7, claim 8 or 9 carrier, antagonist or agonist according to claim 17 evaluation, the antibody of claim 15, the plant of claim 16 or plant tissue, the results material of claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein fine chemicals is a R-Glyceric acid, citromalic acid, fumaric acid, oxysuccinic acid, pyruvic acid, succsinic acid, three hydroxybutyric acids or trihydroxy-butyric acid.

[0554.0.0.16] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.17] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.17] sees [0001.0.0.0]

[0002.0.17.17] γ-An Jidingsuan be used to strengthen specified plant growth, prevent the generation of Powdery Mildew on the grape and suppress some other plant disease.Humans and animals is usually with variable amount picked-up and metabolism γ-An Jidingsuan.γ-An Jidingsuan in 1998 is registered (approval is sold) and is the growth active insecticidal components.γ-An Jidingsuan is important signal, and this signal helps to regulate the operability of mineral substance in plant.The bio-chemical pathway of mineral substance support and control growth and reproduction, and the approach that instructs plant that multiple biology and abiotic stress are reacted.During coercing and some specific period of plant-growth, plant is high especially to the demand of mineral substance.The γ-An Jidingsuan level can improve naturally in these times in the plant.

γ-An Jidingsuan (GABA), nonprotein amino acid accumulates in the plant after being subjected to environmental stimulus usually, and γ-An Jidingsuan accumulation can also causing ethene produces.Exogenous GABA caused as a child about 12 that the ethene production rate was increased to 14 times.GABA causes the active raising of acc synthase mRNA accumulation, ACC level, acc oxidase mRNA level and external acc oxidase.The investigator has proposed GABA as the possible effect of signal transducer, sees Pant Physiol, 115 (1): 129-35 (1997).

γ-An Jidingsuan (GABA), four carbon nonprotein amino acid are the important component in total free aminoacids storehouse in most protokaryons and the eukaryote.In plant, coerce the initiating signal transduction pathway, wherein the kytoplasm Ca of Sheng Gaoing ²⁺Activation Ca ²⁺/ calmodulin relies on the L-Glutamic decarboxylase activity and GABA synthesizes.The H that raises ⁺Also can stimulate the L-Glutamic decarboxylase activity with the substrate level.The GABA accumulation might be mainly to mediate by L-Glutamic decarboxylase.Experimental evidence supports that GABA is synthetic and participates in pH regulator, nitrogen storage, development of plants and defence, and L-glutamic acid utilize can compatible penetrant and alternative pathway, see Trends Pant Sci.4 (11): 446-452 (1999).

γ-An Jidingsuan strengthens root and thereby leaf makes the plant nutrient level be higher than the nutrient level of utilizing single nutrition to obtain to nutraceutical absorption.Coerced and nutrition is taken in when being restricted when plant, should be believed that γ-An Jidingsuan can promote the nutrition utilization, during coercing, strengthened growth thus.

The quick GABA of response to traume is accumulated in have vital role in the plant defense disease and pest (Ramputh and Brown, Plant Physiol.111 (1996): 1349-1352).γ-An Jidingsuan (GABA) has been summarized in Shelp etc., Canadien Journal of Botany (2003) 81,11,1045-1048 as the development of the effective control agent of no spondylopathy insect pest system in the plant.Authors describe available evidence and show that reaction GABA accumulation biological and abiotic stress mediates by the L-Glutamic decarboxylase activation in the plant.Manyly show the GABA block nerves function of absorption and cause the oblique variegated leaf moth larvae of damage as the applied research that suppresses the fact that neurotransmitter works in invertebrate pests based on GABA, and show walk about or by tobacco aphid and tiltedly the food grass of variegated leaf moth larvae stimulate GABA in soybean and tobacco, to accumulate respectively.In addition, the endogenous GABA of elevated levels prevents to be ingested by the tobacco aphid larva and by northern root nodule nematode infections in the tobacco of genetic modification.Therefore, the author reaches a conclusion, and promptly the alternate strategies that farm crop species that high GABA produces potential can be used as chemical insecticide that has of genetic modification is used for handling no spondylopathy insect pest.

Between nursery stage, pollen granule forms conduit and passes female tissue guide micropyle, sends sperm to ovum angiosperm.External, GABA stimulates pollen tube growth.Mouse ear mustard belongs to the transaminase of POP2 genes encoding degraded GABA and helps to form the oriented micropyle, sees Cell.114 (1): 47-59 (2003).

Because these favourable physiological roles of GABA and Agricultural biotechnologies potential need to identify to participate in the metabolic enzyme of GABA or other proteinic gene, and generation have mutant or the transgenic plant strain of utilizing these genetic modifications GABA content.

Shikimic acid is found in various plants.It has two functional groups, optically active hydroxyl and carboxyl in same molecular.They can produce various types of esters and salt.Shikimic acid belongs to the cyclitol class, this means that it is a hydroxylation naphthenic hydrocarbon, comprises at least three oh groups, and each hydroxyl is attached to different ring carbon atoms.Term " shikimic acid " or " shikimin " relate to this compound anionic form and in and attitude.

In fact crucial intermediate was a shikimic acid during all aromatics synthesized in the cell.Aromatics comprises phenylalanine, tyrosine, tryptophane, para-amino benzoic acid and to hydroxybutyric acid.

Glyphosate (N-(phosphonomethyl) glycine) is non-selective broad-spectrum herbicide, and its symplastically is indexed into the meristematic tissue of growing plant.Glyphosate causes EPSP synthase (5-enol pyruvic acid shikimic acid-3-phosphate synthase by suppressing the chloroplast(id) location; EPSP) [EC2.5.1.19] (Amrhein etc., 1980, Pant Physiol.66:830-834) causes the shikimic acid accumulation.

The biosynthetic initial product of most phenolic compounds is a shikimic acid.Phenols because its-the OH group can separate institute and think acidity.Phenols is the compound with reactive behavior, and the short of space restraining effect that is produced by other side chain, and they just can form hydrogen bond.As a result, many flavonoids have intramolecular bond.Another important characteristic is the ability that they and metal form inner complex.They are also oxidized easily, and if oxidized they will form polymkeric substance (deepening gathering).Otch or dying plant part deepening are caused by this reaction.They are inhibited usually to plant-growth.In phenylpropyl alcohol, low-molecular-weight derivant is a large amount of flavouring agents such as tonka bean camphor, styracin, sinapinic acid, lubanol or the like.These materials and derivative thereof also are the biosynthetic intermediates of xylogen simultaneously.

Shikimic acid pathway is connected sugar metabolism with the biosynthesizing of aromatics.In seven metabolism sequence of steps, phosphoenolpyruvic acid and erythrose 4-phosphoric acid are converted into chorismic acid, the precursor of aromatic amino acid and many aromatic series secondary metabolites.All the approach intermediate also is considered to the tapping point compound, and they can be used as the substrate of other pathways metabolism.Shikimic acid pathway only is found in microorganism and the plant, never finds in Mammals.Obtained the pure product of whole enzymes of this approach from protokaryon and eukaryote, and to from several biologies its separately DNA characterize.The cDNA coding of higher plant has the protein of N-terminal signal sequence so that the input plastid shows that plastid is the biosynthetic unique place of chorismic acid.In microorganism, shikimic acid pathway is subjected to the inhibition of checking of the adjusting of feedback inhibition and first enzyme.In higher plant, do not identify the agent of physiology feedback inhibition, show that the approach regulation and control may occur over just gene level.This difference between microorganism and the plant reflects very large variation in the primary structure of first enzyme separately.Several path enzymes exist with the isozyme form, and its expression changes with changing envrionment conditions, and plant, is different from the organ to the organ.This approach penult enzyme is unique target of weedicide glyphosate.Glyphosate tolerance transgenic plant are core (Annual Review of Plant Physiology and Plant MolecularBiology 50 (1999): 473-503) of several crop plants novel herbicidal Controlling System.Term " shikimic acid " or " shikimin " relate to the anionic form and the neutral condition of this compound.

Arrange from the very simple to complexity more according to its complicacy from the natural product of shikimic acid, wherein simple product such as Vanillin (mainly as flavouring agent), Whitfield's ointment (precursor of acetylsalicylic acid), lawsone (naphthoquinones that is used for some opalizer) and eastern gelsemium henbane lactone (once as the ataractic tonka bean camphor in uterus), Fu Za product such as xylan lactone podophyllotoxin more.Podophyllotoxin is to mix the unitary dimer of two phenylpropyl alcohols (from 9 carbon units of shikimic acid) basically.Podophyllotoxin at first obtains from mayapple (Podophyllum peltatum) separation, and mayapple is also referred to as Dysosma versipellis or America Podophyllum emodi var chinense, a kind of plant that is used as catharsis medicine and purgation medicine for a long time.Podophyllotoxin has been used for the treatment of molluscum, and is the mitotic inhibitor that presents anti-tumor activity.Particularly Etoposide is used for the treatment of various forms of lung cancer, carcinoma of testis and acute lymphoblastic leukemia.

Putrescine is by the effect synthetic of healthy cell by ornithine decarboxylase, and putrescine is one of the simplest polyamines and to show be the necessary somatomedin of cell fission.

Experimental evidence shows that polyamines participates in growth, differentiation in plant or form takes place, stress with withered (Evans and Malmberg, 1989).

[0003.0.0.17]％

[0004.0.0.17]％

[0005.0.0.17]％

[0006.0.0.17]％

[0007.0.0.17]％

A kind of method that [0008.0.17.17] improves biosynthesizing throughput is to use recombinant DNA technology.Therefore, people's expectation can produce γ-An Jidingsuan or shikimic acid in plant.The inhuman production of this type allows the most suitable and the most effective quality, quantity and the screening of producing biology of control.The latter is particularly important for commercial production economics, and also is favourable for the human consumer therefore.In addition, people are desirably in the plant and produce γ-An Jidingsuan or shikimic acid, so that improve plant production power and to the resistance of biology as discussed previously and abiotic stress.Term " shikimic acid " or " shikimin " relate to this compound anionic form and in and attitude.

More already used year of recombinant DNA technology method is with by increasing different biosynthesis genes and study the production that its influence to fine chemicals production improves fine chemicals in microorganism and the plant.For example, it is reported that the xenthophylls astaxanthin can result from the nectary of rotaring gene tobacco plant.Those transgenic plant are to prepare by the carrier transformation of tobacco plant that agrobacterium tumefaciens mediates, and wherein carrier comprises the ketolase encoding gene (called after crtO) from H.pluvialis, and the Pds gene of tomato is as the promotor and the leader sequence of encoding.Those results show that about 75% carotenoid that is found in the conversion plant flowers contains ketone group.

[0009.0.17.17] should be favourable if can obtain producing algae, plant or other microorganism of a large amount of γ-An Jidingsuans or shikimic acid or putrescine therefore.The protokaryon or the eukaryotic microorganisms that relate to this type of conversion in some embodiments that the present invention is hereinafter discussed.

If it also should be favourable can obtaining root, leaf, stem, fruit or spend the middle plant that produces a large amount of γ-An Jidingsuans or shikimic acid or putrescine.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

The quality that [0010.0.17.17] therefore improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the γ-An Jidingsuan or the shikimic acid that for example are present in as mentioned above in plant and some microorganisms are limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is specific γ-An Jidingsuan or shikimic acid spectrum in the balanced diet as far as possible, because excessive γ-An Jidingsuan or shikimic acid have negative effect on the specific concentrations.Further improving the quality only may be by adding other γ-An Jidingsuan or shikimic acid, and these compositions all are limited.

[0011.0.17.17] is necessary with balance mode γ-An Jidingsuan or shikimic acid to be added into suitable biology in order to guarantee the high quality of food and animal-feed.

[0012.0.17.17] therefore, still be starved of one or more codings and participate in γ-An Jidingsuan or shikimic acid or the biosynthetic enzyme of putrescine or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but need forever to improve the method that obtains the highest intrinsic energy γ-An Jidingsuan, putrescine and shikimic acid, on the other hand, reduce the byproduct that produces in the production process as far as possible.

[0013.0.0.17] sees [0013.0.0.0]

[0014.0.17.17] therefore in the first embodiment, the present invention relates to produce the method for fine chemicals, and fine chemicals is γ-An Jidingsuan or shikimic acid or putrescine thus.Therefore, in the present invention, term " fine chemicals " refers to γ-An Jidingsuan or shikimic acid or putrescine as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of γ-An Jidingsuan or shikimic acid or putrescine as used herein.

[0015.0.17.17] in one embodiment, term " fine chemicals " or " each fine chemicals " meaning is meant at least a active chemical compound of γ-An Jidingsuan or shikimic acid or putrescine that has.

In one embodiment, term " fine chemicals " meaning is meant γ-An Jidingsuan.In one embodiment, depend on the context that uses it, term " fine chemicals " meaning is meant shikimic acid.In one embodiment, depend on the context that uses it, term " fine chemicals " meaning is meant putrescine.In whole specification sheets, term " fine chemicals " meaning is meant γ-An Jidingsuan or shikimic acid or putrescine, its salt, ester, thioesters or free form or be bonded to other compound such as the form of sugar or glycopolymers (as glucosides, for example bioside).

[0016.0.17.17] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YER156C, YFR042W, YOR084W, YLR375W, b1693, b0651, b0847 or b2965 bak protein; With

(b) in allowing described biology, produce under the condition that fine chemicals is γ-An Jidingsuan, putrescine or shikimic acid and make biological growth.

Therefore, the present invention relates to a kind of method, it may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II A or IIB the 3rd row, 227-230,595-598 capable shown in activity of proteins, perhaps have by Table I A or IB the 5th or 7 row, 227-230,595-598 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce under the condition that fine chemicals is γ-An Jidingsuan, putrescine or shikimic acid and make biological growth.

[0016.1.17.17] therefore, term " fine chemicals " meaning is meant and Table I A or IB 227-229, relevant " γ-An Jidingsuan " or its homologue of the 595 and 596 listed full sequences of row; And the meaning is meant and Table I A or IB the 230th and relevant " shikimic acid " or its homologue of the 597 listed sequences of row; And the meaning is meant and Table I A or relevant " putrescine " of IB the 598th listed sequence of row.

Therefore, term " fine chemicals " can refer to " γ-An Jidingsuan " or " shikimic acid " or " putrescine " according to environment and context.For being meant, the meaning of illustrating term " each fine chemicals " " γ-An Jidingsuan " or " shikimic acid " or " putrescine " from the listed sequence of context also can use term " each fine chemicals ".

[0017.0.0.17] to [0018.0.0.17]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.17.17] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II A or IIB the 3rd row, 227-230, protein active shown in 595-598 is capable or by as Table I A or IB the 5th or 7 row, 227-230, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 595-598 was capable.

[0020.0.17.17] is surprisingly found out that, yeast saccharomyces cerevisiae protein YER156C, YFR042W, YOR084W, YLR375W and/or Escherichia coli protein b1693, b0651, b0847 or b2965 give the γ-An Jidingsuan of the plant that transforms or shikimic acid or putrescine (with regard to described protein and homologue thereof and coding nucleic acid molecule (particularly as shown in Table I A or IB and Table II A or IIB the 3rd row, 227-230,595-598 are capable), being " fine chemicals " or " each fine chemicals ") content at transgene expression in the Arabidopis thaliana to be increased.

[0021.0.0.17] sees [0021.0.0.0]

The sequence of [0022.0.17.17] yeast saccharomyces cerevisiae YER156C has been published in Dietrich, F.S etc., Nature 387 (6632 supplementary issue), 78-81 (1997), and its activity to be defined as be unclassified protein.Therefore, in one embodiment, the inventive method comprise as shown here from yeast saccharomyces cerevisiae YER156C protein or the purposes of the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases γ-An Jidingsuan, the preferred quantity of free or combining form γ-An Jidingsuan.In one embodiment, in the methods of the invention, the activity of unclassified protein YER156C is enhanced.

The sequence of yeast saccharomyces cerevisiae YFR042W has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be the cells survival desired protein.Therefore, in one embodiment, the inventive method comprises the described active proteinic purposes that has as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases γ-An Jidingsuan, the preferred quantity of the γ-An Jidingsuan of free or combining form.In one embodiment, in the methods of the invention, the activity of described cells survival desired protein is enhanced.

The sequence of yeast saccharomyces cerevisiae YOR084W has been published in Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and its activity be defined as be the supposition peroxysome matrix lipase.Therefore, in one embodiment, the inventive method comprises as shown here from the peroxysome matrix lipase of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases γ-An Jidingsuan, the preferred quantity of the γ-An Jidingsuan of free or combining form.In one embodiment, in the methods of the invention, described activity for example peroxysome matrix lipase activity is enhanced.

The sequence of yeast saccharomyces cerevisiae YLR375W has been published in Johnston, M., and Nature 387 (6632 supplementary issue), 87-90 (1997), and its activity is defined as participating in the protein of preceding tRNA montage and branched-chain amino acid absorption.Therefore, in one embodiment, the inventive method comprises as shown here from the preceding tRNA montage of having of yeast saccharomyces cerevisiae and the protein of branched-chain amino acid assimilating activity or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases shikimic acid, the preferred quantity of the shikimic acid of free or combining form.In one embodiment, in the methods of the invention, described activity, the activity of proteins that tRNA montage and branched-chain amino acid absorb before for example participating in is enhanced.

The sequence of e. coli k12 b0651 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be pyrimidine specificity nucleoside hydrolase.Therefore, in one embodiment, the inventive method comprises the purposes with the active protein of pyrimidine specificity nucleoside hydrolase or its homologue from e. coli k12 as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases γ-An Jidingsuan, the preferred quantity of the γ-An Jidingsuan of free or combining form.In one embodiment, in the methods of the invention, described activity for example has the active activity of proteins of pyrimidine specificity nucleoside hydrolase and is enhanced.

The sequence of e. coli k12 b0847 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition translocator.Therefore, in one embodiment, the inventive method comprises as shown here from the active protein of the translocator with supposition of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases γ-An Jidingsuan, the preferred quantity of the γ-An Jidingsuan of free or combining form.In one embodiment, in the methods of the invention, described activity, for example the activity of translocator b0847 is enhanced.

The sequence of e. coli k12 b1693 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the 3-dehydroquinate dehydratase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary 3-dehydroquinate dehydratase protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be shikimic acid or linolic acid and/or comprise shikimic acid, linoleic triglyceride level, lipid, oils and/or fat, particularly increase shikimic acid, linolic acid and/or comprise shikimic acid, linoleic triglyceride level, lipid, oils and/or fat, the preferred linoleic quantity of free or combining form.In one embodiment, 3-dehydroquinate dehydratase activity of proteins is enhanced or produces in the methods of the invention, and for example the activity from colibacillary 3-dehydroquinate dehydratase protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b2965 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the ornithine decarboxylase isozyme.Therefore, in one embodiment, the inventive method comprises as shown here from the protein with ornithine decarboxylase activity of isoenzyme of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases putrescine, the preferred quantity of the putrescine of free or combining form.In one embodiment, in the methods of the invention, described activity, the activity of proteins that for example has ornithine decarboxylase activity is enhanced.

The homologous compound (=homologue) of [0023.0.17.17] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, shown in Table II A or IIB the 3rd row, the 227th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred alpha-aminobutyric acid content increases.

In one embodiment, shown in Table II A or IIB the 3rd row, the 228th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred alpha-aminobutyric acid content increases.

In one embodiment, shown in Table II A or IIB the 3rd row, the 229th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferred alpha-aminobutyric acid content increases.

In one embodiment, shown in Table II A or IIB the 3rd row, the 230th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, shikimic acid content increase in the biology is given in active raising.

In one embodiment, shown in Table II A or IIB the 3rd row, the 595th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, alpha-aminobutyric acid content increase in the biology is given in active raising.

In one embodiment, shown in Table II A or IIB the 3rd row, the 596th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, alpha-aminobutyric acid content increase in the biology is given in active raising.

In one embodiment, shown in Table II A or IIB the 3rd row, the 597th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, shikimic acid content increase in the biology is given in active raising.

In one embodiment, shown in Table II A or IIB the 3rd row, the 598th row in the polypeptide homologue of any one be to have same or similar active homologue.Particularly, putrescine content increase in the biology is given in active raising.

[0023.1.0.17] as the homologue of the polypeptide shown in Table II A or IIB the 3rd row, 227-230,595-598 are capable can be by Table I A or IB the 7th row, 227-230,595-598 capable shown in nucleic acid molecule encoding polypeptide or can be Table II A or IIB the 7th row, 227-230,595-598 capable shown in polypeptide.

The homologue of the polypeptide shown in Table II A or IIB the 3rd row, 227-229,595,596 row can be have increase alpha-aminobutyric acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, 227-229,595,596 row or Table II A or IIB the 7th row, 227-229,595,596 row.

The homologue of the polypeptide shown in Table II A or IIB the 3rd row, the 230th and 597 row can be have increase shikimic acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 230th and 597 row or Table II A or IIB the 7th row, the 230th and 597 row.

The homologue of the polypeptide shown in Table II A or IIB the 3rd row, the 598th row can be have increase putrescine content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 598th row or Table II A or IIB the 7th row, the 598th row.

[0024.0.0.17] sees [0024.0.0.0]

[0025.0.17.17] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause in biology or its part, the preferred described biomass cells γ-An Jidingsuan or shikimic acid or putrescine level to increase, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II A or IIB the 3rd row, 227-229 capable or the 230th the row or 595-598 capable shown in any one proteinic biology or the enzyme activity, if promptly with Table II A or IIB the 3rd row, 227-230, any one yeast saccharomyces cerevisiae protein was compared shown in 595-598 was capable, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I A or IB the 4th row and is expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as increase each fine chemicals in biological or its part.For example play eozoan and express biologically, and play eozoan with biological shown in Table I A or IB the 4th row from identical section, order, guiding principle or door from different sections, order, guiding principle or door.

[0025.1.0.17] sees [0025.1.0.0]

[0026.0.0.17] to [0033.0.0.17]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.17.17] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, its have Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or by Table I A or IB the 5th row, 227-229 is capable or the 230th row or 595-598 capable shown in the protein of nucleic acid molecule encoding or its homologue (as Table I A or IB the 7th row, 227-229 is capable or the 230th row or 595-598 capable shown in homologue) active protein expression level or active aspect difference, and it is difference aspect biological chemistry or genetics reason, and therefore shows each fine chemicals quantity that increases.

[0035.0.0.17] to [0038.0.0.17]: see that [0035.0.0.0] is to [0038.0.0.0]

[0039.0.0.17]: see [0039.0.0.0]

[0040.0.0.17] to [0044.0.0.17]: see that [0040.0.0.0] is to [0044.0.0.0]

[0045.0.17.17] under the situation about being enhanced of yeast saccharomyces cerevisiae protein YER156C or its homologue (shown in Table II A or IIB the 5th or 7 row, the 227th row), give in one embodiment each fine chemicals, preferred γ-An Jidingsuan be increased in 68% and 1029% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YFR042W or its homologue (shown in Table II A or IIB the 5th or 7 row, the 228th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred γ-An Jidingsuan be increased in 91% and 110% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR084W or its homologue, for example under the situation that the activity of Jia Ding peroxysome matrix lipase protein (shown in Table II A or IIB the 5th or 7 row, the 229th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred γ-An Jidingsuan be increased in 71% and 459% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YLR375W or its homologue, under the situation that the activity of the protein that tRNA montage and branched-chain amino acid absorb before for example participating in (shown in Table II A or IIB the 5th or 7 row, the 230th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred shikimic acid be increased in 14% and 26% or more between.

In one embodiment, at Escherichia coli protein b0651 or its homologue, for example under the situation that the activity of the active protein of pyrimidine specificity nucleoside hydrolase (shown in Table II A or IIB the 5th or 7 row, the 595th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred γ-An Jidingsuan be increased in 53% and 206% or more between.

In one embodiment, at Escherichia coli protein b0847 or its homologue, for example have under the situation that the activity of the active protein of translocator (shown in Table II A or IIB the 5th or 7 row, the 596th row) of supposition is enhanced, preferably, give in one embodiment each fine chemicals, preferred γ-An Jidingsuan be increased in 85% and 187% or more between.

In one embodiment, at Escherichia coli protein b1693 or its homologue, for example have under the situation that the activity of the active protein of 3-dehydroquinate dehydratase (shown in Table II A or IIB the 5th or 7 row, the 597th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred shikimic acid be increased in 15% and 31% or more between.

In one embodiment, at Escherichia coli protein b2965 or its homologue, for example have under the situation that the activity of the protein (shown in Table II A or IIB the 5th or 7 row, the 598th row) of ornithine decarboxylase activity is enhanced, preferably, give in one embodiment each fine chemicals, preferred putrescine be increased in 111% and 1693% or more between.

[0046.0.17.17]％

[0047.0.0.17] to [0048.0.0.17]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.17.17] has to give and improves each fine chemicals γ-An Jidingsuan quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:15722 as SEQ ID, 15723,15724,15725,15726,15727 or 15778,15779,91412,91413,91414,91525,91526,91527,91528, consensus sequence shown in 91529 or Table IV the 7th row, the 227th, 228,229,595 and 596 the row shown in or its function homologue described in the literary composition, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 227th, 228,229, nucleic acid molecule or its function homologue as herein described shown in 595 and 596 row) sequence of coded polypeptide, and have the activity that the γ-An Jidingsuan level of giving described in the literary composition increases.

Have to give and improve each fine chemicals shikimic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:15768 as SEQ ID, 15769,15770,15771,91630,91631,91632,91633, (for example Table IV the 7th is listed as consensus sequence shown in 91634, the 230th, shown in 597 row) or Table II A or IIB the 5th or 7 row, the 230th, the sequence of its function homologue described in polypeptide or the literary composition shown in 597 row, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 230th, nucleic acid molecule or its function homologue as herein described shown in 597 row) sequence of coded polypeptide, and have the activity that the shikimic acid level of giving described in the literary composition increases.

Have to give and improve each fine chemicals putrescine quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:91831 as SEQ ID, 91832,91833,91834,91835, (for example Table IV the 7th is listed as consensus sequence shown in 91836, shown in the 598th row) or Table II A or IIB the 5th or 7 row, the sequence of its function homologue described in polypeptide or the literary composition shown in the 598th row, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 598th row) sequence of coded polypeptide, and have the activity that the putrescine level of giving described in the literary composition increases.

[0050.0.17.17] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, the sylvite of γ-An Jidingsuan or shikimic acid or sodium salt or its ester or glucoside (for example its diglucoside).

[0051.0.17.17] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise the composition of γ-An Jidingsuan or shikimic acid or putrescine.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of γ-An Jidingsuan or shikimic acid or putrescine.

[0052.0.0.17] sees [0052.0.0.0]

[0053.0.17.17] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein give nucleic acid molecule encoding of the present invention protein or

Polypeptide of the present invention (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide) express and increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide) express and improve;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide) express and improve;

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide);

(h) by add positive Expression element or remove negative Expression element improve code book invention polypeptide (for example have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.17.17] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having as Table II A or IIB the 3rd or 5 row, protein or its homologue shown in capable or the 230th row of 227-229 or 595-598 are capable (for example Table II A or IIB the 5th or 7 row, polypeptide shown in capable or the 230th row of 227-229 or 595-598 are capable) giving each fine chemicals after active expression of polypeptides or the activity increases.

[0055.0.0.17] to [0067.0.0.17]: see that [0055.0.0.0] is to [0067.0.0.0]

[0068.0.17.17] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of γ-An Jidingsuan or shikimic acid or putrescine.

[0069.0.0.17] sees [0069.0.0.0]

[0070.0.17.17] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example the favourable composition of γ-An Jidingsuan and shikimic acid or their biological chemistry derivative for example contains the γ-An Jidingsuan of high level (from limited physiology of nutrition angle) and the favourable vitamin composition of shikimic acid or putrescine or their derivative.

[0071.0.0.17] sees [0071.0.0.0]

[0072.0.17.17] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds also has their biological chemistry derivative, for example chorismic acid, prephenic acid, anthranilic acid, phenyl-pyruvic acid, phenylalanine, 4-hydroxybenzene butyric acid, tyrosine, tryptophane, Vanillin, Whitfield's ointment, lawsone or eastern gelsemium henbane lactone except shikimic acid.

[0073.0.17.17] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or its homologue (for example Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation.

[0074.0.17.17] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded.

[0075.0.0.17] to [0077.0.0.17]: see that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.17.17] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes.Can use method known to those skilled in the art purified fermentation broth, tunning, plant or plant product.

For these different methods of setting up gradually, product be γ-An Jidingsuan or shikimic acid or putrescine or or also contain the composition of γ-Ding Suan or shikimic acid or putrescine, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.17] to [0084.0.0.17]: see that [0079.0.0.0] is to [0084.0.0.0]

[0084.0.17.17]％

[0085.0.17.17] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.17] to [0087.0.0.17]: see that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.17.17] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Since the nutritive value that for example is used for the plant of raise poultry depend on above-mentioned fine chemicals or plant more antibiont or abiotic stress and output be improved, so this is very important for the plant breeder.

[0088.1.0.17] sees [0088.1.0.0]

[0089.0.0.17] to [0090.0.0.17]: see that [0089.0.0.0] is to [0090.0.0.0]

[0091.0.0.17] sees [0091.0.0.0]

[0092.0.0.17] to [0094.0.0.17]: see that [0092.0.0.0] is to [0094.0.0.0]

[0095.0.17.17] advantageously, by method of the present invention in genetically modified organism, increase γ-An Jidingsuan or shikimic acid or putrescine pond with separate a large amount of pure basically each fine chemicals or be improved to the resistance of biological and abiotic stress and obtain high yield.

[0096.0.17.17] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.

[0097.0.17.17]％

[0098.0.17.17] in preferred embodiments, each fine chemicals be produce according to the present invention and carry out isolating where necessary.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.10.17].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate and comprise each fine chemicals composition.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.17.17] comprises the transgenic plant that the method according to this invention synthetic comprises fine chemicals such as γ-An Jidingsuan or shikimic acid or putrescine and can advantageously directly put on market, and do not need institute's synthetic fine chemicals is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet each fine chemicals that the method according to this invention produces can also be as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free fine chemicals from biology, advantageously separate from plant.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because γ-An Jidingsuan or shikimic acid or putrescine are positioned at cell in the microorganism, must relate to separation to their recovery to biomass.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.

[0101.0.10.17] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.17.17] for example, γ-An Jidingsuan or shikimic acid or putrescine can advantageously detect by HPLC, LC or GC separation method.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect the existence of the product that contains γ-An Jidingsuan or shikimic acid or putrescine to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

[0103.0.17.17] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use have as Table III the 7th row, 227-229 is capable or the 230th row or 595-598 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding schedule IIA or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0104.0.17.17] in one embodiment, nucleic acid molecule of the present invention or process of the present invention or method nucleic acid molecule used therefor and Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th the row or 595-598 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention not by Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide of sequence.

[0105.0.0.17] to [0107.0.0.17]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.17.17] method of the present invention and had Table I A or IB the 5th or 7 row, the nucleic acid molecule of sequence shown in capable or the 230th row of 227-229 or 595-598 are capable, from Table II A or IIB the 5th or 7 row, aminoacid sequence shown in capable or the 230th row of 227-229 or 595-598 are capable is derived or from containing Table IV the 7th row, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in capable or the 230th row of 227-229 or 595-598 are capable, perhaps its coding has as Table II A or IIB the 3rd, 5 or 7 row, the enzymic activity of polypeptide or bioactive polypeptide shown in capable or the 230th row of 227-229 or 595-598 are capable or for example to give each fine chemicals behind its expression or active the increasing be derivative or the homologue that γ-An Jidingsuan or shikimic acid or putrescine increase.

[0109.0.17.17] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.It is synthetic that these nucleic acid constructs make each fine chemicals, particularly γ-An Jidingsuan that the inventive method produced or shikimic acid or putrescine be optimized.

[0110.0.0.17] sees [0110.0.0.0]

[0111.0.0.17] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.17.17] the inventive method is the isolated nucleic acid sequences form, its coding have as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide active polypeptide or have as Table II A or IIB the 5th and 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the polypeptide of peptide sequence, and give γ-An Jidingsuan or shikimic acid or putrescine level and increase.

[0113.0.0.17] to [0114.0.0.17]: see that [0113.0.0.0] is to [0114.0.0.0]

[0115.0.0.17] sees [0115.0.0.0]

[0116.0.0.17] to [0120.0.0.17] sees that [0116.0.0.0] is to [0120.0.0.0]

[0120.1.0.17]：％

[0121.0.17.17] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II A or IIB the 5th or 7 be listed as, peptide sequence or its functional homologue as herein described had the difference of one or more amino acid moleculars shown in capable or the 230th row of 227-229 or 595-598 were capable, described artificial sequence is preferably given aforementioned activity, promptly improving Table II A or IIB the 5th or 7 row, 227-229, giving the γ-An Jidingsuan level after the activity of peptide sequence shown in 595 or 596 row increases, perhaps improving Table II A or IIB the 5th or 7 row, giving the shikimic acid level after the activity of peptide sequence shown in the 230th row or the 597th row increases, and is perhaps improving Table II A or IIB the 5th or 7 row, giving the putrescine level after the activity of peptide sequence shown in the 598th row increases.

[0122.0.0.17] to [0127.0.0.17]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.17.17] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 227-229 is capable or the 230th row or the 595-598 primer shown in capable to) can be based on sequence shown in this paper, for example Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence or from as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence deutero-sequence produce.

[0129.0.17.17] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence was from described comparison shown in Table IV the 7th row, the 227th and 228 row or the 230th row or 595-598 were capable.

[0130.0.17.17] can use degenerated primer to have the new protein fragment of aforementioned activity (giving γ-An Jidingsuan or shikimic acid increase as comprise this segmental protein expression or activity in raising after) by pcr amplification then.

[0131.0.0.17] to [0138.0.0.17]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.17.17] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with for the Table I A of γ-An Jidingsuan or IB the 5th or 7 row, 227-229,595,596 row or for the Table I A of shikimic acid or IB the 5th or 7 row, the 230th, 597 row or for the Table I A of putrescine or IB the 5th or 7 row, sequence hybridization shown in the 598th row, and the coding expression has each fine chemicals, the i.e. active peptide of γ-An Jidingsuan or shikimic acid or putrescine of increasing.

[0140.0.0.17] to [0146.0.0.17]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.17.17] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.17.17] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I A or IB the 5th or 7 row, nucleotide sequence or its portion homologous were at least about 30% shown in capable or the 230th row of 227-229 or 595-598 were capable, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases γ-An Jidingsuan or shikimic acid or putrescine.

[0149.0.17.17] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in one of nucleotide sequence or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding have above-mentioned activity (increasing) as giving γ-An Jidingsuan or shikimic acid or putrescine and randomly as Table II A or IIB the 5th row, 227-229 is capable or the 230th row or 595-598 capable shown in the protein of protein active.

[00149.1.0.17] randomly, in one embodiment, with Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or the 595-598 protein shown in capable is known activity or is used for these protein of note.

[0150.0.17.17] in addition, nucleic acid molecule of the present invention can only contain Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example giving γ-An Jidingsuan or shikimic acid or putrescine when its active raising increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in one of sequence sense strand, Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 227-229 is capable or the 230th row or 595-598 capable shown in the right PCR of primer will produce as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th capable or 595-598 capable shown in fragment or its gene product of polynucleotide sequence.

[0151.0.0.17]: see [0151.0.0.0]

[0152.0.17.17] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising γ-An Jidingsuan as be shown in the examples (227-229,595,596 row) or shikimic acid (the 230th, 597 row) or putrescine (the 598th row).

[0153.0.17.17] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in protein its part has Table II A for example as herein described or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in the activity of polypeptide.

[0154.0.17.17] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.17] to [0156.0.0.17]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.17.17] the present invention relates in addition owing to the genetic code degeneracy is different from Table I A or IB the 5th or 7 row, one of nucleotide sequence (with its part) shown in capable or the 230th row of 227-229 or 595-598 are capable and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 5th or 7 row, the polypeptide of consensus sequence shown in capable or the 230th row of 227-229 or 595-598 are capable or as Table II A or IIB the 5th or 7 be listed as, the nucleic acid molecule of polypeptide or its function homologue shown in capable or the 230th row of 227-229 or 595-598 are capable.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment as Table IV the 5th or 7 row, 227-229 is capable or the 230th row or the consensus sequence of 595-598 shown in capable or as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 5th or 7 row, 227-229 is capable or the 230th row or the consensus sequence of 595-598 shown in capable or as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable.

[0158.0.0.17] to [0160.0.0.17]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.17.17] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.17] sees [0162.0.0.0]

[0163.0.17.17] preferably, under stringent condition with Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given in biology or its part for example each fine chemicals quantity increase in the compartment of tissue, cell or cell after using activity of proteins).

[0164.0.0.17] sees [0164.0.0.0]

[0165.0.17.17] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.17] to [0167.0.0.17]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.17.17] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 aminoacid sequence shown in capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II A or IIB the 5th or 7 be listed as, capable or the 230th row of 227-229 or the sequence of 595-598 shown in capable are identical at least about 60%, more preferably with as Table II A or IIB the 5th or 7 be listed as, 227-229 is capable or one of the 230th row or 595-598 sequence shown in capable are identical at least about 70%, even more preferably with as Table II A or IIB the 5th or 7 be listed as, capable or the 230th row of 227-229 or the sequence of 595-598 shown in capable are at least about 80%, 90% or 95% homology, and most preferably with as Table II A or IIB the 5th or 7 be listed as, capable or the 230th row of 227-229 or the sequence of 595-598 shown in capable are at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.17] to [0172.0.0.17]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.17.17] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:15720 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:15720 sequence before use.

[0174.0.0.17]: see [0174.0.0.0]

[0175.0.17.17] for example, the sequence that has 80% homology at protein level and SEQ ID NO:15721 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:15721 sequence.

[0176.0.17.17] is by replacing, insert or lack from being listed as Table II A or IIB the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in capable or the 230th row of 227-229 or 595-598 were capable be listed as according to of the present invention as Table II A or IIB the 5th or 7, one of polypeptide had at least 30% shown in capable or the 230th row of 227-229 or 595-598 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II A or IIB the 5th or 7 row, polypeptide had essentially identical character and discerns shown in capable or the 230th row of 227-229 or 595-598 were capable.

[0177.0.17.17] is by replacing, insert or lack from being listed as Table I A or IB the 5th or 7 according to of the present invention, the functional equivalent that obtains of nucleotide sequence shown in capable or the 230th row of 227-229 or 595-598 are capable be listed as according to of the present invention as Table II A or IIB the 5th or 7, one of polypeptide had at least 30% shown in capable or the 230th row of 227-229 or 595-598 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II A or IIB the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in capable or the 230th row of 227-229 or 595-598 are capable.

[0178.0.0.17] sees [0178.0.0.0]

[0179.0.17.17] can by to the nucleotide sequence of nucleic acid molecule of the present invention (particularly as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in) in introduce replacement, interpolation or the disappearance of one or more Nucleotide, and thereby to introduce in the coded protein one or more amino acid replace, add or disappearance and produce coding as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th go or 595-598 capable shown in the nucleic acid molecule of homologue of protein sequence.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 introduce sudden change in the encoding sequence of sequence shown in capable.

[0180.0.0.17] to [0183.0.0.17]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.17.17] is employed to have as Table I A or IB the 5th or 7 row, the nucleotide sequence homologous compound of sequence shown in capable or the 230th row of 227-229 or 595-598 are capable, perhaps come Table II A or IIB the 5th or 7 row freely, the homologous compound of nucleotide sequence of sequence shown in capable or the 230th row of 227-229 or 595-598 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.17.17] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 arbitrary sequence shown in capable in other Nucleotide of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.17.17] also preferred the inventive method comprise as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence identical.

[0187.0.17.17] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprise as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.17.17] still has the basic the enzyme activity of polypeptide of the present invention (being its active basic reduction that do not have) of giving each fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in and under the same conditions the polypeptide expressed activity compare and do not reduce substantially.

[0189.0.17.17] as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the homologous compound of sequence, or deutero-as Table II A or IIB the 5th or 7 row, 227-229 is capable or the homologue of the 230th row or the 595-598 sequence shown in capable also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.17]: see [0190.0.0.0]

Biological or its part of [0191.0.17.17] and described contrast or selection is compared, should biology or its part produces free and/or each fine chemicals level of bonded improves according to the inventive method described herein.

[0192.0.0.17] to [0203.0.0.17]: see that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.17.17] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly γ-An Jidingsuan (227-229,595,596 row) or shikimic acid (the 230th, 597 row) or putrescine (the 598th row) quantity increase;

(b) comprise, preferably comprise mature form at least as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595th, 596 row or the 597th row or the 598th row shown in nucleic acid molecule or its segmental nucleic acid molecule, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 5th or 7 row, 227-229 is capable or the 230th row or the 595th capable, the nucleic acid molecule that primer shown in the 596 or the 597th row or the 598th row or primer obtain amplification from cDNA library or genomic library, and in biological or its part, give each fine chemicals, i.e. the increase of γ-An Jidingsuan (227-229,595,596 row) or shikimic acid (the 230th, 597 row) or putrescine (the 598th row) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 5th or 7 row, 227-229 is capable or the 230th row shown in consensus sequence and in biological or its part, give each fine chemicals, the i.e. nucleic acid molecule that increases of γ-An Jidingsuan (227-229,595,596 row) or shikimic acid (the 230th, 597 row) or putrescine (the 598th row) quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be the nucleic acid molecule that γ-An Jidingsuan (227-229,595,596 row) or shikimic acid (the 230th, 597 row) or putrescine (the 598th row) quantity increase, described peptide coding as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row shown in the structural domain of polypeptide; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I A or IB the 5th or 7, capable or the 230th row of 227-229 or the nucleic acid molecule of 595-598 shown in capable or coding (optimized encoding is mature form at least) are as Table II A or IIB the 5th or 7 row, at least the 15nt of nucleic acid molecule of polypeptide shown in capable or the 230th row of 227-229 or 595-598 are capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby, the nucleic acid molecule of preferred (a) to (l) by one or more Nucleotide be different from as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable.In one embodiment, nucleic acid molecule not by Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable is formed.In one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 peptide sequence shown in capable.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide have the different polypeptide of one or more amino acid at least and therefore do not encode as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the protein of sequence.Therefore, in one embodiment, by nucleotide sequence (a)-(l) encoded protein matter not by Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in protein sequence at least 30% identical and with Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% identical.

[0205.0.0.17] to [0206.0.0.17]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.17.17] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of glutamic acid metabolism, phosphoenolpyruvic acid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.17] to [0226.0.0.17]: see that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.17.17] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the capable sequence or derivatives thereof of mentioning of 595-598, can advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one L-glutamic acid or phosphoenolpyruvic acid pathways metabolism in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.17.17] therefore cultivated to cross simultaneously in another embodiment of the present invention and expressed coding and the biology that directly or indirectly participates in L-glutamic acid or metabolic proteinic at least one nucleic acid of phosphoenolpyruvic acid or a gene.

[0229.0.17.17] can with present method employed as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence and/or the combination of aforementioned biosynthesis gene other favourable nucleotide sequences of expressing be the sequence of other gene of coding aromatic amino acid (as tryptophane, phenylalanine or tyrosine) approach.These genes cause the synthetic increase of essential amino acids tryptophan, phenylalanine or tyrosine.

[0230.0.0.17] sees [230.0.0.0]

[0231.0.17.17] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded γ-An Jidingsuan or shikimic acid simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.Those skilled in the art are known to quantity biological, that particularly suppressing in the plant must the aromatic amino acid tryptophane, tyrosine or phenylalanine biosynthetic enzyme or gene can increase shikimic acid.In addition, the shikimic acid that can cause accumulating in the biology to the inhibition of enzyme of degraded tryptophane, phenylalanine or tyrosine increases.

[0232.0.0.17] to [0276.0.0.17]: see that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.17.17] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.

[0278.0.0.17] to [0282.0.0.17]: see that [0278.0.0.0] is to [0282.0.0.0]

[0283.0.17.17] in addition, can from cell, separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in protein, perhaps as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the antibody of polypeptide, it can utilize polypeptide of the present invention or its fragment to produce by standard technique.Monoclonal antibody preferably.

[0284.0.0.17] sees [0284.0.0.0]

[0285.0.17.17] in one embodiment, the present invention relates to have as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence or by as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.17.17] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in capable or the 230th row of 227-229 or 595-598 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence or by its polypeptide of forming shown in capable or the 230th row of 227-229 or 595-598 are capable, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to and comprising more than a polypeptide as consensus sequence (each row) shown in Table IV the 7th row, 227-230 and/or 595-598 are capable.

[0287.0.0.17] to [0289.0.0.17]: see that [0287.0.0.0] is to [0289.0.0.0]

[00290.0.17.17] uses Vector NTI Suite 8.0, (InforMax ^TM, Invitrogen ^TMLifescience software, U.S.Main Office, 7305 Executive Way, Frederick, MD21704, U.S.) assembly AlignX software (on September 25th, 2002) and use following setting to compare: for the pairing comparison: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Right for multiple ratio: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Breach separates the point penalty scope: 8; Residue is replaced matrix: blosum62; Hydrophilic residue: G P S N D Q E K R; Conversion weight: 0.5; Total calculation options: total residue mark: 0.9.Also selected to set in advance in order to compare conserved amino acid.

[0291.0.17.17] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 sequence shown in capable.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 227-229 capable or the 230th row or the sequence of 595-598 shown in capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 227-229 capable or the 230th row or the sequence of 595-598 shown in capable.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence form.

[0292.0.0.17] sees [0292.0.0.0]

[0293.0.17.17] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in other sequence of sequence phase region.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence form.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.17.17] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, the polypeptide of activity of proteins shown in capable or the 230th row of 227-229 or 595-598 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, 227-229 capable or the 230th the row or 595-598 capable shown in sequence.

[0295.0.0.17] to [0296.0.0.17]: see that [0295.0.0.0] is to [0296.0.0.0]

[0297.0.0.17] sees [0297.0.0.0]

[00297.1.0.17]％

[0298.0.17.17] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the identical aminoacid sequence of sequence.

[0299.0.17.17] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in one of nucleotide sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.17.17] is therefore, and be described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th capable or 595-598 capable shown in sequence different.Therefore, this polypeptide contain with as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the complete amino acid sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.17] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.17.17] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II A or IIB the 5th or 7 row, 227-229 capable or the 230th row or the aminoacid sequence of 595-598 shown in capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.17] sees [0303.0.0.0]

[0304.0.17.17] operation nucleic acid molecule of the present invention may cause generation have basically as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.17] to [0308.0.0.17]: see that [0305.0.0.0] is to [0308.0.0.0]

[0309.0.17.17] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II A or IIB the 5th or 7 row, capable or the 230th row of 227-229 or the protein of 595-598 shown in capable are meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, and be not shown in Table II A or IIB the 5th or 7 row, capable or the 230th row of 227-229 or " other polypeptide " of 595-598 in capable are meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II A or IIB the 5th or 7 row, polypeptide shown in capable or the 230th row of 227-229 or 595-598 are capable is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II A or IIB the 3rd row, capable or the 230th row of 227-229 or 595-598 are proteinic and from the protein of identical or different biology shown in capable.In one embodiment, be not shown in Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or capable " other polypeptide " of 595-598 are not given in biology or its part each fine chemicals and increased.

[0310.0.0.17] to [0334.0.0.17]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.17.17] confirmed the dsRNAi method to reduce as Table I A or IB the 5th or 7 row, 227-229 is capable or the expression of the 230th row or the 595-598 nucleotide sequence shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.17] to [0342.0.0.17]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.17.17] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.17] to [0350.0.0.17]: see that [0344.0.0.0] is to [0350.0.0.0]

[0351.0.0.17] to [0361.0.0.17]: see that [0351.0.0.0] is to [0361.0.0.0]

[0362.0.17.17] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II A or IIB the 5th or 7 row, 227-229 capable or the 230th row or the polypeptide of 595-598 shown in capable, for example coding has protein such as Table II A or IIB the 3rd row, the polypeptide of polypeptide active shown in capable or the 230th row of 227-229 or 595-598 are capable) nucleic acid molecule.

Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Have as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide (for example have as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

[0363.0.0.17] sees [0363.0.0.0]

[0364.0.17.17] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---for example naturally occurring coding as Table II A or IIB the 3rd row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide of the present invention gene promotor with accordingly as Table I A or IB the 5th row, 227-229 is capable or the 230th row or 595-598 capable shown in the combination of protein coding sequence---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.17] to[0373.0.0.17]: see that [0365.0.0.0] is to [0373.0.0.0]

[0374.0.17.17] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, γ-An Jidingsuan that produces in the inventive method or shikimic acid, particularly each fine chemicals also can be from plant with free γ-An Jidingsuan or shikimic acid, particularly each fine chemicals or be bonded to compound or the isolated in form of part (moiety) (for example glucosides such as bioside).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.17] to[0376.0.0.17]: see that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.17.17] therefore the invention still further relates to the method according to this invention, separated the γ-An Jidingsuan or the shikimic acid that are produced thus.

[0378.0.17.17] can separate by this way and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than 90% the γ-An Jidingsuan or the shikimic acid that produce in the method.As required, resulting γ-An Jidingsuan or shikimic acid can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.17.17] in one embodiment, γ-An Jidingsuan and shikimic acid are the mixtures of each fine chemicals.

The suitable synthetic parent material of γ-An Jidingsuan that [0380.0.17.17] obtains in the method for the invention or shikimic acid as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises γ-An Jidingsuan that separation produces or shikimic acid composition or each fine chemicals (if expectation) that is produced, but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention be the γ-An Jidingsuan that produces of the inventive method or shikimic acid or genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine purposes or be used to produce the purposes of γ-An Jidingsuan or shikimic acid, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

[0381.0.0.17] to [0382.0.0.17]: see that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.17.17]

[0384.0.0.17] sees [0384.0.0.0]

The fermented liquid that [0385.0.17.17] obtains with this kind approach particularly contains the fermented liquid with other organic acid, amino acid, polypeptide or polysaccharide blended γ-An Jidingsuan or shikimic acid, and dry matter content accounts for the 1-70% of weight, preferred 7.5 to 25% usually.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means and to utilize the concentration of sugar during this period of time to remain on or be reduced to 0-10g/l in the fermention medium, preferred to 0-3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.17.17] therefore can also be further purified the γ-An Jidingsuan or the shikimic acid that produce according to the present invention.For this reason, the composition that will contain product separates (by for example open column chromatography or HPLC), and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.17] to [0392.0.0.17]: see that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.17.17] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 nucleic acid molecule shown in capable is hybridized under lax stringent condition nucleic acid molecule, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.17] to [0398.0.0.17]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.17.17] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving each fine chemicals generation raising under these conditions.

[00399.1.0.17] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing whether this effect depends on as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or the 595-598 polypeptide shown in capable or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.17] to [0415.0.0.17]: see that [0400.0.0.0] is to [0415.0.0.0]

[0416.0.0.17] sees [0416.0.0.0]

[0417.0.17.17] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor to γ-An Jidingsuan or shikimic acid or putrescine biosynthetic pathway has resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block γ-An Jidingsuan or shikimic acid or putrescine synthetic inhibitor.

[0418.0.0.17] to [0423.0.0.17]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.17.17] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other organic acids.

Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.17] to [0434.0.0.0]: see that [0425.0.0.0] is to [0434.0.0.0]

[0435.0.17.17] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.17.17] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce γ-An Jidingsuan or shikimic acid or putrescine.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.17.17] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.17.17] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.17.17] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.17.17] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad 50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.17] sees [0441.0.0.0]

[0442.0.0.17] sees [0442.0.0.0]

[0443.0.0.17] sees [0443.0.0.0]

[0444.0.17.17] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.17.17] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.17] to [0450.0.0.17]: see that [0446.0.0.0] is to [0450.0.0.0]

[0451.0.0.17] sees [0451.0.5.5]

[0452.0.0.17] to [0453.0.0.17]: see that [0452.0.0.0] is to [0453.0.0.0]

[0454.0.17.17] embodiment 8: analyzing nucleic acid molecules is to the influence of γ-An Jidingsuan or shikimic acid or putrescine generation

[0455.0.17.17] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example γ-An Jidingsuan or shikimic acid or putrescine) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being γ-An Jidingsuan or shikimic acid or putrescine) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLCin Biochemistry " in (1987) " Laboratory Techniques inBiochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing forBiotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biological Materials ", John Wiley andSons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia ofIndustrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation andpurification techniques in biotechnology ", Noyes Publications).

[0456.0.0.17]: see [0456.0.0.0]

[0457.0.17.17] embodiment 9: γ-An Jidingsuan or shikimic acid or putrescine purifying

[0458.0.17.17] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS or TLC can clearly detect the existence of γ-An Jidingsuan or shikimic acid or putrescine product to the biology analysis of recombinating.The biological total quantity that yeast produced that for example is used for the inventive method can be according to the following step analysis: by ultrasonic, the grinding of glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed such as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 402 2S1V diluters with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.17ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, Hewlett Packard 1100 HPLC have been used, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Technologies, can be from Ultra Scientific Inc., 250Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6 * 250mm[Waters, 34 Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

[0459.0.17.17] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified γ-An Jidingsuan or shikimic acid or putrescine.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.0.17] sees [0460.0.0.0]

[0461.0.17.17] embodiment 10: clone SEQ ID NO:15720,15762,15708,15712,91094,91415,91530 or 91635 is used for expressing plant

[0462.0.0.17] sees [0462.0.0.0]

[0463.0.17.17] passes through pcr amplification SEQ ID NO:15720,15762,15708,15712,91094,91415,91530 or 91635 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.0.17] to [0466.0.0.17]: see that [0464.0.0.0] is to [0466.0.0.0]

[0466.1.0.17] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.17.17] selects following primer sequence for genes of SEQ ID NO:15720:

I) forward primer (SEQ ID NO:15760)

atgaatagcg?taaaaagagt?aaagct

Ii) reverse primer (SEQ ID NO:15761)

ctaggccaaa?gacatcttag?cca

Select following primer sequence for genes of SEQ ID NO:15762:

I) forward primer (SEQ ID NO:15766)

atggcaggta?tcaagttgac?gcat

Ii) reverse primer (SEQ ID NO:15767)

tcattttgtt?aatagttttt?tgtatgct

Select following primer sequence for genes of SEQ ID NO:15708:

I) forward primer (SEQ ID NO:15710)

atggaacaga?acaggttcaa?gaaag

Ii) reverse primer (SEQ ID NO:15711)

ttacagtttt?tgtttagtcg?ttttaac

Select following primer sequence for genes of SEQ ID NO:15712:

I) forward primer (SEQ ID NO:15718)

atgagtaatg?caaacaatag?tgctat

Ii) reverse primer (SEQ ID NO:15719)

tcaatggtat?ttatagccgc?attgt

Selection be used to clone as Table III the 5th row, 227-230 is capable or 595-598 capable shown in more primers of gene be described in Table III the 7th row, 227-230 is capable or 595-598 is capable.

[0468.0.17.17] to [0470.0.17.17]: see that [0468.0.0.0] is to [0470.0.0.0]

The PCR product that [0470.1.17.17] utilizes the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBIFermentas) phosphorylation to produce by Pfu Turbo archaeal dna polymerase, and the clone enters in the binary vector of treated mistake.

[0471.0.17.17] sees [0471.0.0.0]

[0471.1.17.17] uses Pfu Turbo archaeal dna polymerase benefit flat in the second step building-up reactions by the DNA end of the PCR product that the Herculase archaeal dna polymerase produces.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Utilize the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBI Fermentas) phosphorylation PCR product, and be cloned in the carrier of handling.

[0472.0.17.17] to [0479.0.17.17]: see that [0472.0.0.0] is to [0479.0.0.0]

[0480.0.17.17] embodiment 11: express the generation of SEQ ID NO:15720,15762,15708,15712,91094,91415,91530 or 91635 transgenic plant

[0481.0.0.17] to [0513.0.0.17]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.17.17] alternatively, as Haak and Reineke, Antimicrob.AgentsChemother.19 (3): detect γ-An Jidingsuan described in 493 (1981).

Alternatively, as Gould and Erickson, J Antibiot 41 (5), and 688-9 detects shikimic acid described in (1988).

Alternatively, as Endo Y., detect putrescine described in Anal Biochem.89 (1): the 235-46 (1978).

The different plants of being analyzed the results are shown in following table 1:

Table 1

??ORF	Metabolite	Method	??min	??max
					??YER156C	γ-An Jidingsuan	??GC	??1.68	??11.29
??YFR042W	γ-An Jidingsuan	??GC	??1.91	??2.10
					??YOR084W	γ-An Jidingsuan	??GC	??1.71	??5.59
??YLR375W	Shikimic acid	??GC	??1.14	??1.26

??ORF	Metabolite	Method	??min	??max
					??b0651	γ-An Jidingsuan (GABA)	??GC	??1.53	??3.06
??b1693	Shikimic acid	??GC	??1.15	??1.31
					??b2965	Putrescine	??GC	??2.11	??17.93
??b0847	γ-An Jidingsuan (GABA)	??GC	??1.85	??2.87

[0515.0.0.17] to [0552.0.0.17]: see that [0515.0.0.0] is to [0552.0.0.0]

[0552.1.0.17]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

?ORF	Metabolite	??Min	??Max
				?YLR375W	Shikimic acid	??2.12	??5.11

Table 2 shows that shikimic acid has increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YLR375W.

At yeast saccharomyces cerevisiae protein YLR375W or have participate in before under the activity of the protein of tRNA montage and branched-chain amino acid assimilating activity or its homologue situation about being enhanced, preferably, give the fine chemicals shikimic acid and be increased between 112% and 411%.

[00552.2.0.17]：see[00552.2.0.0]

[0553.0.17.17]

1. produce the method for γ-An Jidingsuan or shikimic acid or putrescine, it comprises:

A) improve in non-human being or its one or more parts or produce as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in the activity of protein or its function equivalent; With

B) in allowing described biology, cultivate this biology under the condition of generation γ-An Jidingsuan or shikimic acid or putrescine.

2. produce the method for γ-An Jidingsuan or shikimic acid or putrescine, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part γ-An Jidingsuan or shikimic acid or putrescine quantity to be increased;

B) contain just like Table I A or IB the 5th or 7 row, 227-229 is capable or the nucleic acid molecule of the 230th row or the 595-598 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of γ-An Jidingsuan or shikimic acid or putrescine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of γ-An Jidingsuan or shikimic acid or putrescine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that γ-An Jidingsuan in biology or its part or shikimic acid or putrescine quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give in biology or its part γ-An Jidingsuan or shikimic acid or putrescine quantity and increase;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part γ-An Jidingsuan or shikimic acid or putrescine quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 227-229 is capable or the 230th row or 595-598 capable shown in consensus sequence polypeptide and give biology or its part in γ-An Jidingsuan or shikimic acid or putrescine quantity increase; With

I) nucleic acid molecule, its can by under stringent hybridization condition with containing nucleic acid molecule (a) extremely

The suitable nucleic acid library of probe of one of sequence (k) or the screening of its fragment obtains, and give γ-An Jidingsuan or shikimic acid or the increase of putrescine quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded γ-An Jidingsuan or shikimic acid or putrescine.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded γ-An Jidingsuan or the shikimic acid or the putrescine of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding as Table II A or IIB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part γ-An Jidingsuan or shikimic acid or putrescine quantity to be increased;

B) contain just like Table I A or IB the 5th or 7 row, 227-229 is capable or the nucleic acid molecule of the 230th row or the 595-598 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of γ-An Jidingsuan or shikimic acid or putrescine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of γ-An Jidingsuan or shikimic acid or putrescine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that γ-An Jidingsuan in biology or its part or shikimic acid or putrescine quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th row, 227-229 is capable or the 230th row or 595-598 capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give in biology or its part γ-An Jidingsuan or shikimic acid or putrescine quantity and increase;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part γ-An Jidingsuan or shikimic acid or putrescine quantity and increase;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, 227-229 is capable or the 230th row or 595-598 capable shown in consensus sequence polypeptide and give biology or its part in γ-An Jidingsuan or shikimic acid or putrescine quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give γ-An Jidingsuan or shikimic acid or the increase of putrescine quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A or IB the 5th or 7 row, 227-229 is capable or the 230th row or 595-598 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding as claim 6 described in, thus this polypeptide by one or more amino acid be different from as Table II A or IIB the 5th or 7 be listed as, 227-229 is capable or the 230th capable or 595-598 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in γ-An Jidingsuan or shikimic acid or putrescine quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part γ-An Jidingsuan or shikimic acid or putrescine quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps γ-An Jidingsuan or shikimic acid or putrescine level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the γ-An Jidingsuan that will measure or shikimic acid or putrescine level or expression of polypeptides level and the standard γ-An Jidingsuan of when described candidate compound or the sample that comprises described multiple compound lack, measuring or shikimic acid or putrescine or expression of polypeptides level; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that γ-An Jidingsuan in plant or the microorganism or shikimic acid or putrescine produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of γ-An Jidingsuan or shikimic acid or putrescine quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of expression of polypeptides of γ-An Jidingsuan in biology or its part or shikimic acid or the increase of putrescine quantity and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that γ-An Jidingsuan in the cell or shikimic acid or putrescine produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that γ-An Jidingsuan or shikimic acid or putrescine increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce γ-An Jidingsuan or shikimic acid or putrescine;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) γ-An Jidingsuan or shikimic acid or the putrescine level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give γ-An Jidingsuan or shikimic acid or the raising of putrescine level in the host cell after it is expressed with wild-type.

20. identify and to give the method that γ-An Jidingsuan in the cell or shikimic acid or putrescine produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that γ-An Jidingsuan in biology or its part or shikimic acid or putrescine quantity or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce γ-An Jidingsuan or shikimic acid or putrescine;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) γ-An Jidingsuan or shikimic acid or the putrescine level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give γ-An Jidingsuan or shikimic acid or the raising of putrescine level in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that γ-An Jidingsuan or shikimic acid or putrescine increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control γ-An Jidingsuan or shikimic acid or putrescine level at biology.

25. agrochemicals, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of any one described method of claim 1-5, claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein fine chemicals is γ-An Jidingsuan or shikimic acid or putrescine.

[0554.0.0.17] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.18] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.18.18] the present invention relates to produce the method for fine chemicals in microorganism, vegetable cell, plant, plant tissue or its one or more parts.In addition, the invention still further relates to nucleic acid molecule, polypeptide, nucleic acid construct, carrier, antisense molecule, antibody, host cell, plant tissue, reproductive material, results material, plant, microorganism, and Pestcidal compositions and their purposes.

[0002.0.18.18] coenzyme is the molecule that participates in enzyme catalysis.As enzyme, coenzyme is modulation during catalysis; They or unmodified or can regenerated.Every type coenzyme has specific chemical functional.Coenzyme can be attached to certain enzyme or free existing in solution by covalent linkage, but all participates in enzymatic chemical reaction closely with arbitrary mode coenzyme.

[0003.0.18.18] Coenzyme Q10 99.0 (CoQ 10) or ubiquinone are VITAMIN or vitamin-like material in essence.Although there is arguement in name, VITAMIN is defined as essential a small amount of organic compound that the precursor as coenzyme or coenzyme works in the normal body function.Coenzyme Q10 99.0 or CoQ10 belong to the material family that is called ubiquinone.Ubiquinone also is known as ubiquinone and ubiquinone, is lipophilic water-insoluble substance, and the electron transport and the energy that participate in the plastosome produce.The basic structure of ubiquinone comprises benzoquinones " head " and terpene " afterbody "." head " structure participates in the redox active of electron transport chain.Difference main between the various ubiquinones is the quantity of isoprenoid unit (5-carbon structure) in " afterbody ".Ubiquinone comprises 1-12 isoprenoid unit at " afterbody "; Common 10 isoprenoid unit in animal.Ubiquinone is present in most aerobionts, from the bacterium to the plant and animal.There are two cover coding schemes in name for isoprenoid element number in the terpene " afterbody ": ubiquinone n and ubiquinone (x).N is meant the number of isoprenoid side chain, and x is meant the number of carbon in the terpene " afterbody " and can be any multiple of 5.Therefore, Coenzyme Q10 99.0 is meant and has 10 unitary ubiquinones of isoprenoid in " afterbody ".Since each isoprenoid unit has 5 carbon, Coenzyme Q10 99.0 also can called after ubiquinone (50).The structure of ubiquinone and the similar of multiprenylmenaquinone.Coenzyme Q10 99.0 also is known as ubiquinone (50), CoQ10, CoQ (50), ubiquinone (50), ubiquinol-10 and ubidecarerone.

They are natural to be present in the food and also synthetic in vivo sometimes.Same CoQ10 is to be found in the various foods on a small quantity and all to synthesize in the tissue.Biosynthesizing from the amino acid tyrosine to CoQ10 is the rapid process of multistep, needs at least 8 kinds of VITAMIN and several trace element.Coenzyme is a cofactor, and quite a large amount of and complicated enzyme definitely relies on its function.Coenzyme Q10 99.0 is the coenzyme of the enzyme in the other parts at least three kinds of cyclophorases (composite I, II and III) and the cell.The cyclophorase of oxidative phosphorylation approach is for energy-rich phosphate, Triphosaden (ATP) (all cell functions depend on Triphosaden), generation be essential.The electronics of quinone ring and proton transfer function all are very important for all biological; As the ubiquinone in the animal mitochondria, plastoquinone in the plant chloroplast and the vitamin k4 in the bacterium.Term " bioenergetics " has been used to describe biochemical field, and the special concern cellular energy produces.In the free-radical chemistry association area, discovered CoQ10 with its reduction form as good antioxidant.The bioenergetics of CoQ10 and free-radical chemistry are summarized in the book Energy andDefense of the Gian Paolo Littarru of publication in 1994.The precise chemical structure structure of CoQ10 is 2,3 dimethoxys-5 methyl-60 an isopentene group benzoquinones.

[0004.0.18.18] since nineteen fifty-seven was found, CoQ-10 also was called ubiquinone, because it belongs to a compounds that is called quinone, also because it is prevalent in the life biology, was present in heart, liver and kidney especially.CoQ-10 has keying action in the production capacity process in cell.And CoQ-10 or good antioxidant mean the cell injury molecule that it helps to neutralize and is called free radical.Owing to carry out processing and manufacturing by whole cells in vivo, CoQ-10 also finds to be present on a small quantity in the food, particularly in meat and the fish.In middle 1970s, set up the industrial technology of producing pure CoQ10 so that obtain the amount that is enough to be used in more clinical experiments.CoQ10 mainly can separate from microorganism or plant or algae and obtains; Particularly plastosome is the common source of CoQ10.Alternatively, they can obtain from animal or fish easily.

[0005.0.18.18] because the effect of the CoQ10 that replenishes is not also illustrated, still there are a large amount of suppositions in the mechanism of these effects.Yet, much be known about the biochemical characteristic of CoQ10.CoQ10 is a cofactor essential in the plastosome electron transport chain, and CoQ10 accepts the electronics from complex body I and II in electron transport chain, and it is vital that this activity produces for ATP.CoQ10 has antioxidant activity in plastosome He on the cytolemma, prevent the peroxidation of lipid film.CoQ10 also suppresses the oxygenizement of LDL-cholesterol.LDL-cholesterol oxygenizement has vital role in incidence of atherosclerosis mechanism.CoQ10 is biosynthetic in vivo and has the common route of synthesis with cholesterol.

The CoQ10 level reduces along with aging in human body.Why such situation taking place remain unknown, but may be because the reduction that takes place along with aging synthetic and/or the fat peroxidation that improves.Have been noted that in the diversified disease that in animal and human's body of being studied the CoQ10 level significantly reduces.CoQ10 lacks excessively to be utilized by body by insufficient diet CoQ10, CoQ10 biosynthesizing defective, CoQ10 and cause, or united by these three kinds of effects and to cause.In chronic malnutrition and emaciation, infer it to be that the diet absorption that reduces causes.

[0006.0.18.18] studying the Relative Contribution of CoQ10 biosynthesizing to diet CoQ10.Karl Folkers thinks that the main source of the interior CoQ10 of human body is biosynthesizing.This kind mixture is synthetic by 17 step procedure, need at least 7 kinds of VITAMIN (Wei ShengsuB2-riboflavin, vitamin B3-niacinamide, vitamin B6, folic acid, vitamin B12, vitamins C and pantothenic acid) and several trace element, from its character, this mixture is very unsettled.Karl Folkers proof human Central Asia best nutritional thing take in almost be very general and subsequently the second stage can cause damage to the CoQ10 biosynthesizing.This average or " normally " level that will mean CoQ10 is actually the suboptimal level, then only represents the top that lacks iceberg in the low-down level that seriously ill state observation arrives.

The CoQ10 that replenishes can have Cardioprotective activity, cell protection activity and neuroprotective activity.The CoQ10 that statement here replenishes has active effect in cancer, muscular dystrophy and immunodeficiency disease.Equally, it can also suppress fat or strengthen exercise performance.

[0007.0.18.18] is used for blocking the CoQ10 biosynthesizing equally by the HMG-CoA reductase enzyme of the cholesterol levels of blocking-up cholesterol biosynthesizing processing rising.The blood CoQ10 level of resulting reduction is because local CoQ10 of sharing and cholesterol biosynthetic pathway.This is not only a kind of laboratory observation result in suffering from the patient of cardiac failure.It has significant deleterious effect, and this deleterious effect can be eliminated by oral CoQ10 additive.

The CoQ10 body consumption that raises is to infer it is the inducement of low blood CoQ10 level seen under overtired, hypermetabolism and acute apoplexy state.Might operate three kinds of mechanism (the CoQ10 deficiency of ingesting, the CoQ10 biosynthesizing is impaired and the excessive utilization of CoQ10) changes in the overwhelming majority and observes the degree that CoQ10 lacks.

[0008.0.18.18] also found coenzyme Q-10-Q9 at occurring in nature.For example Q9 is the derivative of the CoQ10 that finds in plant chloroplast.Q9 has the shorter aliphatic group that is bonded to ring structure.Because the structural homology of height, coenzyme Q-10-Q9 expection can provide the activity identical or closely similar with CoQ10 in cell or biology.Yet, Matsura etc., Biochim Biophys Acta, 1992,1123 (3), the 309-15 page or leaf is reached a conclusion from its research, and promptly CoQ9 continues in liver cell as the potential antioxidant, and CoQ10 mainly presents antioxidant activity in the cell of CoQ10 as main CoQ homologue.Coenzyme Q10 99.0 is actually in the dissimilar makeup very common composition, and this is because the provide protection of Coenzyme Q10 99.0 Green Tea Extract and the predictive role in the skin tension thereof.

[0009.0.18.18] therefore, coenzyme particularly CoQ10 or CoQ9 can be used in a large amount of different application, for example is used for makeup, medicine and feed and food.

[0010.0.18.18] therefore improves the productivity of described coenzyme and the quality of raising makeup, medicine, food and animal-feed, particularly nutritional additive is a task important in the different industry.

[0011.0.18.18] must operate the natural biological of coenzyme described in the described biology is synthetic in order to guarantee the high productivity of described coenzyme in plant or microorganism.

[0012.0.18.18] therefore, still be starved of one or more codings and participate in the biosynthetic enzyme of described coenzyme or other proteinic suitable gene, and make and not form unwanted byproduct with specific some the described coenzyme of generation of technical scale.Be used for the screening of biosynthetic gene, two features all are even more important.On the one hand, but need forever to improve the method that obtains the described coenzyme of the highest intrinsic energy, on the other hand, reduce the byproduct that produces in the production process as far as possible.

[0013.0.0.18] finds now by providing the embodiment that characterizes in method of the present invention as herein described and the claim can realize this purpose.

[0014.0.18.18] has been found that express the excessively Coenzyme Q10 99.0 or the Q9 content of giving plant of the nucleic acid molecule that this paper characterizes increase.Therefore, in the first embodiment, the present invention relates to produce the method for Coenzyme Q10 99.0 and/or Q9.Therefore, in another embodiment, the present invention relates to produce the method for fine chemicals, fine chemicals is Coenzyme Q10 99.0 and/or Q9 thus.Therefore, in the present invention, term " fine chemicals " refers to " Coenzyme Q10 99.0 and/or Q9 " as used herein.In addition, in another embodiment, term " fine chemicals " also refers to comprise the fine chemicals composition of Coenzyme Q10 99.0 and/or Q9 as used herein.

[0015.0.18.18] depends on context environmental in one embodiment, and term " each fine chemicals " meaning is meant Coenzyme Q10 99.0 and/or Q9.For example, the increase with the gene product of gene of locus/ORF seat title YPR138C, YBR184W, b2699 or b1829 or its homologue of being mentioned gives that coenzyme CoQ10 level increases in plant or its part.For example, the increase with the gene product of gene of locus/ORF seat title YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 or b0175 or its homologue of being mentioned gives that coenzyme CoQ9 level increases in plant or its part.For example, have and give from the increase of the gene product of the gene of colibacillary ORF seat title b2699 or its homologue that coenzyme CoQ9 and CoQ10 level increase in plant or its part.For example, give as the raising of sequence shown in table the 231-234 is capable that coenzyme CoQ10 level increases in plant or its part.For example, give as the raising of sequence shown in table the 235-242 and/or 599-602 are capable that coenzyme CoQ9 level increases in plant or its part.

Therefore, in one embodiment, term " each fine chemicals " meaning is meant Coenzyme Q10 99.0 and Q9.In one embodiment, term " each fine chemicals " meaning is meant Coenzyme Q10 99.0 or Q9.In another embodiment, term " each fine chemicals " meaning is meant Coenzyme Q10 99.0 and/or Q9, with and salt, ester or thioesters, or free form or be bonded to protein (for example enzyme or peptide such as polypeptide) or be bonded to the Coenzyme Q10 99.0 and/or the Q9 of film or its part, for example with the form of oil or wax or with the form of the composition of lipid, oils, fat or lipid mixtures, and the Coenzyme Q10 99.0 of reduction or oxidised form and/or Q9.In preferred embodiments, term " each fine chemicals " meaning is meant free form or its salt form or is bonded to peptide or the Coenzyme Q10 99.0 of protein form or CoQ9.Lipid, oils, wax, fat or lipid mixtures refer to comprise any lipid, oils, wax and/or the fat of any combination or free Coenzyme Q10 99.0 and/or Q9.

In one embodiment, term " fine chemicals " and term " each fine chemicals " meaning is meant to have the active at least a chemical compound of above-mentioned fine chemicals.

[0016.0.18.18] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YPR138C, YBR184W, b2699 and/or b1829 protein; With

(b) in allowing described biology, produce each fine chemicals, for example CoQ10 or comprise under the condition of composition of each fine chemicals and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein; With

(b) in allowing described biology, produce each fine chemicals, for example CoQ9 or comprise under the condition of composition of each fine chemicals and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II the 3rd row, 231-234 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, 231-234 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) allowing to produce fine chemicals, for example make biological growth under the condition of CoQ10.Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II the 3rd row, 235-242 and/or 599-602 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, 235-242 and/or 599-602 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) allowing to produce fine chemicals, for example make biological growth under the condition of CoQ9.

[0016.1.0.18] in one embodiment, the inventive method is given more than one each fine chemicals, promptly Q9 and/or Coenzyme Q10 99.0 content increase.

Therefore, according to environment and context, term term " fine chemicals " can refer to " Q9 " and/or " Coenzyme Q10 99.0 ".Be meant " Q9 " and/or " Coenzyme Q10 99.0 " in order to illustrate term " fine chemicals " meaning, can also use term " each fine chemicals ".

[0017.0.0.18] to [0018.0.0.18] sees that [0017.0.0.0] is to [0018.0.0.0]

[0019.0.18.18] the inventive method advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " with following defined reference the meaning make finger for example with such biophase ratio, promptly should not exist YPR138C by biology, YBR184W, b2699, b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, the b0175 activity of proteins is carried out above-mentioned modification.

[0020.0.18.18] is surprisingly found out that, at least a yeast saccharomyces cerevisiae protein YPR138C or YBR184W or e. coli k12 protein b2699 or b1829 transgene expression in Arabidopis thaliana are given the CoQ10 content increase in the plant that transforms.

Be surprisingly found out that at least a yeast saccharomyces cerevisiae protein YPR172W, YER174C, YER156C or YDR513W or e. coli k12 protein b2426, b2703, b2729, b3644, b3605, b2699, b0730 or b0175 transgene expression in Arabidopis thaliana are given the CoQ9 content increase in the plant that transforms.

Be surprisingly found out that e. coli k12 protein b2699 transgene expression in Arabidopis thaliana is given CoQ9 and the increase of CoQ10 content in the plant of transforming.

[0021.0.0.18] sees [0021.0.0.0]

The sequence (accession number NP 416921) of [0022.0.18.18] e. coli k12 b2426 has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the oxydo-reductase with NAD (P) binding domains.Therefore, in one embodiment, the inventive method comprises as shown here having " ribitol dehydrogenase; short-chain alcohol dehydrogenase homology " active gene product of superfamily from colibacillary, be preferably and participate in C-compound and carbohydrate utilization, VITAMIN, cofactor and prothetic group metabolism, come from the biosynthesizing of the secondary species of primary amino acid, aerobic aromatic series metabolism, metabolism of anaerobism aromatic series and/or secondary metabolites metabolism, halfcystine-aromatic series biosynthesizing and/or halfcystine-die aromatischen Aminosaeuren degraded, the aromatic series anabolism, the protein or its homologue that more preferably have the oxidoreductase activity with NAD (P) binding domains of supposition (for example are listed as this paper Table II the 5th or 7, shown in the 599th row or by Table I the 5th or 7 row, nucleic acid molecule encoding shown in the 599th row) purposes, it is used for producing fine chemicals in biological or its part as described, be Q9, particularly increase Q9, the quantity of the Q9 of preferred free or combining form.

The sequence of e. coli k12 b2729 (accession number YP 026181) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be hydrogenase expression/formation protein.Therefore, in one embodiment, the inventive method comprises as shown here having " hydrogenase expression/formation protein hypD "-active gene product of superfamily from colibacillary, being preferably protein or its homologue with hydrogenase expression/formation protein active (for example is listed as this paper Table II the 5th or 7, shown in the 601st row or by Table I the 5th or 7 row, nucleic acid molecule encoding shown in the 601st row) purposes, it is used for producing fine chemicals in biological or its part as described, be Q9, particularly increase Q9, the quantity of the Q9 of preferred free or combining form.

The sequence of e. coli k12 b2703 (accession number NP 417209) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be phosphotransferase system enzyme II; Sorbyl alcohol/Sorbitol Powder specificity.Therefore, in one embodiment, the inventive method comprises as shown here having " phosphotransferase system Sorbitol Powder specific enzymes II; factor II "-active gene product of superfamily from colibacillary, be preferably and participate in plasma membrane, cell input, phosphorylation and dephosphorylation modifications, C-compound, carbohydrate transhipment, sugar combination and/or phosphotransferase system, preferred have a phosphotransferase system enzyme II; The protein of sorbyl alcohol/Sorbitol Powder activity specific or its homologue (for example as this paper Table II the 5th or 7 row, the 600th the row shown in or by Table I the 5th or 7 row, the 600th the row shown in nucleic acid molecule encoding) purposes, it is used for producing fine chemicals in biological or its part as described, be Q9, particularly increase Q9, the preferred quantity of the Q9 of free or combining form.

The sequence of e. coli k12 b3644 (accession number NP 418101) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the protein of the stress-induced that do not characterize.Therefore, in one embodiment, the inventive method comprises the protein HI0467 superfamily protein from colibacillary supposition as shown here, the purposes of the protein of preferred stress-induced or its homologue (for example shown in this paper Table II the 5th or 7 row, the 602nd row or) by nucleic acid molecule encoding shown in Table I the 5th or 7 row, the 602nd row, it is used for producing fine chemicals in biological or its part as described, be Q9, the preferred Q9 of free or combining form, particularly increase Q9, the preferred quantity of the Q9 of free or combining form.In one embodiment, in the methods of the invention, the activity of proteins of the stress-induced of Biao Zhenging is not enhanced or produces, and for example is enhanced or produces from the protein of colibacillary stress-induced or the activity of its homologue.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be DNA chain exchange and the recombinant protein with proteolytic enzyme and nuclease.Therefore, in one embodiment, the inventive method comprises recombinant protein recA superfamily protein as shown here, particularly having DNA reorganization and DNA repairs, the pheromone reaction, the mating type decision, sex-specific protein, the protein of Nucleotide combination and/or proteolytic enzyme and nuclease, particularly have the DNA chain exchange of proteolytic enzyme and nuclease and recombinant protein or its homologue (for example as this paper Table II the 5th or 7 row, shown in the 233rd row or by Table I the 5th or 7 row, nucleic acid molecule encoding shown in the 233rd row) purposes, it is used for producing fine chemicals in biological or its part as described, i.e. the Coenzyme Q10 99.0 of reduction or oxidation, preferred free form or the reduction of combining form or derivative form or the Coenzyme Q10 99.0 of oxidation.In one embodiment, in the methods of the invention, protein with proteolytic enzyme and nuclease, particularly have the DNA chain exchange of recombinant protein rec A superfamily proteolytic enzyme and nuclease and the activity of recombinant protein or its homologue and be enhanced or produce, for example have the DNA chain exchange of recombinant protein rec A superfamily proteolytic enzyme and nuclease and the activity of recombinant protein or its homologue is enhanced or produces from colibacillary.

The sequence of e. coli k12 b1829 (accession number NP_416343) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the heat shock protein with protease activity.Therefore, in one embodiment, the inventive method comprises the reaction of coercing that has as shown here, the pheromone reaction, the mating type decision, protein modification or have the protein of proteolytic activity or for the proteinic purposes of sex-specific, proteolytic enzyme particularly, the heat shock protein that particularly has protease activity, preferred heat shock protein htpX superfamily or its homologue are (for example as this paper Table II the 5th or 7 row, shown in the 234th row or by Table I the 5th or 7 row, nucleic acid molecule encoding shown in the 234th row) purposes, it is used for producing fine chemicals in biological or its part as described, the i.e. reduction or the Coenzyme Q10 99.0 of oxidation particularly increase the quantity of the Coenzyme Q10 99.0 of the reduction of free or combining form or oxidation.In one embodiment, in the methods of the invention, proteolytic enzyme, particularly have the heat shock protein of protease activity, the activity of preferred heat shock protein htpX superfamily is enhanced or produces, for example the activity that from colibacillary proteolytic enzyme, particularly has the heat shock protein of protease activity, preferred heat shock protein htpX superfamily or its homologue is enhanced or produces.

The sequence of e. coli k12 b3605 (accession number NP_418062) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the L-serum lactic dehydrogenase.Therefore, in one embodiment, the inventive method comprises (S)-2-hydroxy acid oxidase homology superfamily protein as shown here, particularly have in lipid, during breathing, lipid acid and isoprenoid metabolism be used as electronics/active protein of hydrogen vehicle and/or in lipid, has active protein in lipid acid and the isoprenoid degraded, for example L-serum lactic dehydrogenase or its homologue are (for example as this paper Table II the 5th or 7 row, shown in the 239th row or by Table I the 5th or 7 row, nucleic acid molecule encoding shown in the 239th row) purposes, it is used for producing fine chemicals in biological or its part as described, i.e. the Q9 of reduction or oxidation, preferred free form or the reduction of combining form or derivative form or the Q9 of oxidation.In one embodiment, in the methods of the invention, (S)-2-hydroxy acid oxidase homology superfamily protein, particularly having the protein of L-lactate dehydrogenase activity or the activity of its homologue is enhanced or produces, for example, particularly have the protein of L-lactate dehydrogenase activity or the activity of its homologue and be enhanced or produce from colibacillary (S)-2-hydroxy acid oxidase homology superfamily protein.

The sequence of e. coli k12 b2699 (accession number NP_417179) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be DNA chain exchange and the recombinant protein with proteolytic enzyme and nuclease.Therefore, in one embodiment, the inventive method comprises recombinant protein recA superfamily protein as shown here, particularly having DNA reorganization and DNA repairs, the pheromone reaction, the mating type decision, sex-specific protein, the protein of Nucleotide combination and/or proteolytic enzyme and nuclease, particularly have the DNA chain exchange of proteolytic enzyme and nuclease and recombinant protein or its homologue (for example as this paper Table II the 5th or 7 row, shown in the 240th row or by Table I the 5th or 7 row, nucleic acid molecule encoding shown in the 240th row) purposes, it is used for producing fine chemicals in biological or its part as described, i.e. the Q9 of reduction or oxidation, preferred free form or the reduction of combining form or derivative form or the Q9 of oxidation.In one embodiment, in the methods of the invention, have the protein of proteolytic enzyme and nuclease, particularly have the DNA chain exchange of recombinant protein rec A superfamily proteolytic enzyme and nuclease and the activity of recombinant protein or its homologue and be enhanced or produce, for example from colibacillaryly having the protein of proteolytic enzyme and nuclease, particularly having the DNA chain exchange of recombinant protein rec A superfamily proteolytic enzyme and nuclease and the activity of recombinant protein or its homologue and be enhanced or produce.

The sequence of e. coli k12 b0730 (accession number NP_415258) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be that succinic thiokinase operon transcriptional and acyl are replied regulatory gene.Therefore, in one embodiment, the inventive method comprises transcriptional GntR superfamily protein as shown here, particularly having C-compound and carbohydrate utilization regulates, transcribe control, the protokaryon nucleoid, transcription repressor, succinic thiokinase operon transcriptional DNA combination or lipid acid are replied the active protein of regulatory gene, for example succinic thiokinase operon transcriptional and acyl are replied regulatory gene or its homologue (for example is listed as this paper Table II the 5th or 7, shown in the 241st row or by Table I the 5th or 7 row, nucleic acid molecule encoding shown in the 241st row) purposes, it is used for producing fine chemicals in biological or its part as described, i.e. the Q9 of reduction or oxidation, preferred free form or the reduction of combining form or derivative form or the Q9 of oxidation.In one embodiment, in the methods of the invention, the activity that succinic thiokinase operon transcriptional and acyl are replied regulatory gene protein or its homologue is enhanced or produces, and for example the activity of replying regulatory gene protein or its homologue from colibacillary succinic thiokinase operon transcriptional and acyl is enhanced or produces.

The sequence of e. coli k12 b0175 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be CDP-diester glycerine synthase.Therefore; in one embodiment; the inventive method comprises as shown here having a nucleotide metabolism from colibacillary; the phosphatide biosynthesizing; lipid; lipid acid or isoprenoid metabolism; cell communication; the protein of signal transduction or cell sense and reactive behavior; preferred phosphatidic acid two cytidine acyltransferase superfamily protein; preferred CDP-diester glycerine synthase or its homologue are (for example as this paper Table II the 5th or 7 row; shown in the 242nd row or by Table I the 5th or 7 row; nucleic acid molecule encoding shown in the 242nd row) purposes; it is used for producing fine chemicals in biological or its part as described, i.e. the Q9 of reduction or oxidation; preferred free or the reduction of combining form or the Q9 of oxidation.In one embodiment; in the methods of the invention; described activity; phosphatidic acid two cytidine acyltransferase activities for example; particularly the activity of CDP-diester glycerine synthase is enhanced or produces; for example from colibacillary phosphatidic acid two cytidine acyltransferase activities, particularly the activity of CDP-diester glycerine synthase or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YPR138C (accession number NP_015464) has been published in Bussey, H etc., Nature 387 (6632 supplementary issue), 103-105 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be characterized as being be NH4 ⁺Transporter.Therefore, in one embodiment, the inventive method comprises NH4 as shown here ⁺Transporter or ammonium transporter or ammonium transporter nrgA superfamily proteinic purposes, particularly protein have anion transport body (Cl, SO ₄, PO ₄Deng), other cation transfer body (Na, K, Ca, NH ⁴Deng), the transhipment of nitrogen and sulphur, cell input and/or the active proteinic purposes of plasma membrane, it is used to produce Coenzyme Q10 99.0.Therefore, in one embodiment, the inventive method comprises the purposes of yeast saccharomyces cerevisiae YPR138C (for example shown in this paper Table II the 231st row, the 3rd or 5 row) or its homologous compound (for example shown in this paper Table II the 231st row, the 7th row), it is used for producing each fine chemicals in biological or its part as described, be Coenzyme Q10 99.0, particularly increase the quantity of the Coenzyme Q10 99.0 of the reduction of the Coenzyme Q10 99.0 of reduction and/or oxidation, preferably free or combining form and/or oxidation.In one embodiment, in the methods of the invention, NH4 ⁺The activity of transporter is enhanced or produces, for example from the NH4 of yeast saccharomyces cerevisiae ⁺The activity of transporter or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YBR184W (accession number NP_009743) has been published in Feldmann, and H. etc., EMBO are (24) J.13,5795-5809 (1994) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not identified yet and is characterized.Therefore, in one embodiment, the inventive method comprises the purposes of yeast saccharomyces cerevisiae YBR184W (for example shown in this paper Table II the 232nd row, the 3rd or 5 row) or its homologous compound (for example shown in this paper Table II the 232nd row, the 7th row), it is used for producing each fine chemicals, being Coenzyme Q10 99.0 in biological or its part, particularly increases the Coenzyme Q10 99.0 of reduction and/or oxidation, the preferred quantity of the Coenzyme Q10 99.0 of free or combining form.In one embodiment, in the methods of the invention, the activity of yeast saccharomyces cerevisiae YBR 184W or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YPR172W (accession number NP 015498) has been published in Bussey, H. etc., Nature 387 (6632 supplementary issue), 103-105 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not identified yet and is characterized.Therefore, in one embodiment, the inventive method comprises the purposes of yeast saccharomyces cerevisiae YPR172W (for example shown in this paper Table II the 235th row, the 3rd or 5 row) or its homologous compound (for example shown in this paper Table II the 235th row, the 7th row), it is used for producing each fine chemicals, being Q9 in biological or its part, particularly increases the Q9 of reduction and/or oxidation, the preferred quantity of the Q9 of free or combining form.In one embodiment, the activity of yeast saccharomyces cerevisiae YPR172W or its homologue is enhanced or produces in the methods of the invention.

The sequence of yeast saccharomyces cerevisiae YER174C (accession number NP_011101) has been published in Dietrich, F.S etc., Nature 387 (6632 supplementary issue), 78-81 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be characterized as being be the reactive gsh dependency of hydrogen peroxide and superoxide radical oxydo-reductase.Therefore, in one embodiment, the inventive method comprises reactive gsh dependency oxydo-reductase of hydrogen peroxide and superoxide radical or Trx homology superfamily protein, and the protein of for example have protein modification, coercing reactive behavior is used to produce the purposes of Q9.Therefore, in one embodiment, the inventive method comprises the purposes of yeast saccharomyces cerevisiae YER174C (for example shown in this paper Table II the 236th row, the 3rd or 5 row) or its homologous compound (for example shown in this paper Table II the 236th row, the 7th row), it is used for producing each fine chemicals, being Q9 in biological or its part, particularly increases reduction and/or the Q9 of oxidation and/or the quantity of Q9 of free or combining form.In one embodiment, in the methods of the invention, the activity of the reactive gsh dependency of hydrogen peroxide and superoxide radical oxydo-reductase is enhanced or produces, and for example is enhanced or produces from the hydrogen peroxide of yeast saccharomyces cerevisiae and the activity of the reactive gsh dependency oxydo-reductase of superoxide radical or its homologue.

The sequence of yeast saccharomyces cerevisiae YER156C (accession number NP_011083) has been published in Dietrich etc., Nature 387 (6632 supplementary issue), 78-81 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not identified yet and is characterized.Therefore, in one embodiment, the inventive method comprises that the arabidopsis thaliana protein F2K15.180 superfamily protein of YER156C or supposition is used to produce the purposes of Q9.Therefore, in one embodiment, the inventive method comprises the purposes of yeast saccharomyces cerevisiae YER156C (for example shown in this paper Table II the 237th row, the 3rd or 5 row) or its homologous compound (for example shown in this paper Table II the 237th row, the 7th row), it is used for producing each fine chemicals, being Q9 in biological or its part, particularly increases the Q9 of reduction and/or oxidation, the preferred quantity of the Q9 of free or combining form.In one embodiment, the activity of yeast saccharomyces cerevisiae YER156C or its homologue is enhanced or produces in the methods of the invention.

The sequence of yeast saccharomyces cerevisiae YDR513W (accession number NP_010801) has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive to be characterized as being be glutathione reductase.Therefore, in one embodiment, the inventive method comprises glutathione reductase or glutaredoxin superfamily protein, particularly have deoxyribonucleotide metabolism, cytoplasm, coerce reaction, detoxify, and/or the electron transport energy relevant with film preserved the purposes that active protein is used to produce Q9.Therefore, in one embodiment, the inventive method comprises the purposes of yeast saccharomyces cerevisiae YDR513W (for example shown in this paper Table II the 238th row, the 3rd or 5 row) or its homologous compound (for example shown in this paper Table II the 238th row, the 7th row), it is used for producing each fine chemicals, being Q9 in biological or its part, particularly increases reduction and/or the Q9 of oxidation and/or the quantity of Q9 of free or combining form.In one embodiment, in the methods of the invention, the activity of glutathione reductase is enhanced or produces, and for example is enhanced or produces from the glutathione reductase of yeast saccharomyces cerevisiae or the activity of its homologue.

The homologous compound (=homologue) of [0023.0.18.18] invention gene product can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.

Yet, it be known to those skilled in the art that active and (if known words) that homologue preferably has a described increase fine chemicals in biology with protein shown at least a Table II the 3rd row, for example have by the protein that contains the coded peptide sequence of nucleotide sequence of sequence shown in Table I the 5th or 7 row and have identical biological function or activity.

In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in the polypeptide homologue of any one be as described herein or note have a same or similar active homologue.Particularly, each fine chemicals content increase in the biology is given in active raising.In one embodiment, homologue be have Table I or II the 7th row, the 231st or 232 or 235-238 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in any one homologue of polypeptide from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in any one homologue of polypeptide from Ascomycota.In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in any one homologue of polypeptide from yeast.In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in any one homologue of polypeptide from the yeast guiding principle.In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in any one homologue of polypeptide be homologue from Saccharomycetes.In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in any one homologue of polypeptide be to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, the 231st or 232 or 235-238 capable shown in any one homologue of polypeptide be to have same or similar activity (Coenzyme Q10 99.0 in biology or its part is given in particularly active raising and/or Q9 content increases) and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the polypeptide homologue of any one be as described herein or note have a same or similar active homologue.Particularly, each fine chemicals content increase in the biology is given in active raising.In one embodiment, homologue be have Table I or II the 7th row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the polypeptide homologue of any one be derived from bacterium.In one embodiment, Table II the 3rd row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the polypeptide homologue of any one be derived from Proteobacteria.In one embodiment, Table II the 3rd row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the polypeptide homologue of any one be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the polypeptide any one homologue from the enterobacteria order.In one embodiment, Table II the 3rd row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the polypeptide homologue of any one be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, the 233rd or 234 or 239-242 and/or 599-602 capable shown in the polypeptide homologue of any one be to have same or similar activity (Coenzyme Q10 99.0 in biology or its part is given in particularly active raising and/or Q9 content increases) and from the homologue of Escherichia.

The homologue of polypeptide shown in [0023.1.18.18] Table II the 3rd row, 231-234 are capable can be have increase Coenzyme Q10 99.0 content and/or quantity is active, by Table I the 7th row, 231-234 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 231-234 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, 235-242 and/or 599-602 are capable can be have increase Q9 content and/or quantity is active, by Table I the 7th row, 235-242 and/or 599-602 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 235-242 and/or 599-602 capable shown in polypeptide.

[0024.0.0.18] sees [0024.0.0.0]

[0025.0.18.18] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause biology or its part, each fine chemicals in the preferred described biomass cells (particularly Coenzyme Q10 99.0 and/or Q9) and/or comprise the polypeptide of Coenzyme Q10 99.0 and/or Q9, protein, peptide, enzyme, triacylglycerol, lipid, oils, wax, film, membrane portions (membrane fraction) and/or fat level increase, and then described protein or polypeptide have " YPR138C; YBR184W; b2699 and/or b1829 and/or YPR172W; YER174C; YER156C; YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 activity of proteins ".In one embodiment, protein has as activity of proteins shown in Table II the 3rd or 5 row, 231-234 and/or 235-242 and/or 599-602 are capable.In whole specification sheets, in preferred embodiments, if this kind protein or polypeptide still have Table II the 3rd or 5 row, proteinic biology or the enzyme activity shown in 231-234 and/or 235-242 and/or 599-602 are capable, if promptly with yeast saccharomyces cerevisiae or colibacillary YPR138C, YBR184W, b2699, and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein or its combination are compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.3.18.18] in one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and is expressed in evolution with in the eozoan biology far away, the used polypeptide of polypeptide of the present invention or the inventive method is still given described activity, as increase each fine chemicals in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

[0025.2.0.18] sees [0025.2.0.0]

[0025.1.0.18] sees [0025.1.0.0]

[0026.0.0.18] to [0033.0.0.18] sees that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.18.18] preferably, reference, contrast or wild-type are only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention or the used polypeptide of the inventive method, these differences are owing to for example nucleic acid molecule of the present invention or the raising of the inventive method nucleic acid molecule used therefor level, or due to the raising of the specific activity of polypeptide of the present invention or the used polypeptide of the inventive method.For example, its have Table II the 3rd row, 231-234 and/or 235-242 and/or 599-602 capable shown in protein or by Table I the 5th row, 231-234 and/or 235-242 and/or 599-602 capable shown in the protein of nucleic acid molecule encoding or its homologue (as homologue shown in Table I the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable) active protein expression level or active aspect difference, and they are different and show each fine chemicals quantity that increases aspect biological chemistry or genetics reason.

[0035.0.0.18] to [0044.0.0.18] sees that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.18.18] in one embodiment, under the situation that the activity of e. coli k12 protein b2426 or its homologue (shown in Table II the 5th or 7 row, the 599th row) is enhanced, for example have under the situation that the activity of the oxydo-reductase of NAD (P) binding domains is enhanced, preferably give each fine chemicals, preferred Q9 be increased in 38% and 77% or more between.

In one embodiment, at e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of the protein of the stress-induced that does not characterize (for example shown in Table II the 5th or 7 row, the 602nd row) is enhanced, preferably give fine chemicals, preferred Q9 be increased in 37% and 130% or more between.

In one embodiment, at e. coli k12 protein b2703 or its homologue, for example under the activity situation about being enhanced of sorbyl alcohol/Sorbitol Powder specificity phosphotransferase system enzyme II (for example shown in Table II the 5th or 7 row, the 600th row), preferably, give fine chemicals, preferred Q9 be increased in 37% and 118% or more between.

In one embodiment, at e. coli k12 protein b2729 or its homologue, for example under the activity situation about being enhanced of hydrogenase expression/formation protein (for example shown in Table II the 5th or 7 row, the 601st row), preferably, give fine chemicals, preferred Q9 be increased in 35% and 41% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, for example has the reaction of coercing, the pheromone reaction, the mating type decision, active protein of protein modification or proteolytic degradation or sex-specific protein, proteolytic enzyme particularly, the heat shock protein that particularly has protease activity, preferred heat shock protein htpX superfamily or its homologue are (as Table II the 5th or 7 row, shown in the 234th row) activity situation about being enhanced under, give each fine chemicals, preferred Coenzyme Q10 99.0 be increased in 48% and 433% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example have DNA reorganization and DNA repairing activity, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, particularly recombinant protein recA superfamily is (for example as Table II the 5th or 7 row, shown in the 233rd row) activity situation about being enhanced under, give each fine chemicals, preferred Coenzyme Q10 99.0 be increased in 62% and 220% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example have DNA reorganization and DNA repairing activity, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, particularly recombinant protein recA superfamily is (for example as Table II the 5th or 7 row, shown in the 240th row) activity situation about being enhanced under, give each fine chemicals, preferred Q9 be increased in 34% and 253% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example have DNA reorganization and DNA repairing activity, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, particularly recombinant protein recA superfamily is (for example as Table II the 5th or 7 row, shown in the 233rd and 240 row) activity situation about being enhanced under, preferably give each fine chemicals, between preferred Coenzyme Q10 99.0 is increased in 62% and 220% and Q9 be increased in 34% and 253% or more between.

In one embodiment, at e. coli k12 protein b3605 or its homologue, (S)-2-hydroxy acid oxidase homology superfamily protein for example, particularly have in lipid, during breathing, lipid acid and isoprenoid metabolism be used as electronics/active protein of hydrogen vehicle and/or in lipid, has active protein in lipid acid and the isoprenoid degraded, for example the L-serum lactic dehydrogenase is (for example as Table II the 5th or 7 row, shown in the 239th row) activity situation about being enhanced under, preferably give each fine chemicals, preferred Q9 be increased in 35% and 41% or more between.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example protein succinic thiokinase operon transcriptional and acyl are replied under the activity situation about being enhanced of regulatory gene (for example shown in Table II the 5th or 7 row, the 241st row), preferably give each fine chemicals, preferred Q9 be increased in 30% and 203% or more between.

In one embodiment; at e. coli k12 protein b0175 or its homologue; for example in nucleotide metabolism, phosphatide biosynthesizing, lipid, lipid acid or isoprenoid metabolism, cell communication, signal transduction or cell sense and reaction, has active protein; preferred phosphatidic acid two cytidine acyltransferase superfamily protein; under the situation that the activity of preferred CDP-diester glycerine synthase (for example shown in Table II the 5th or 7 row, the 242nd row) is enhanced, preferably give each fine chemicals, preferred Q9 be increased in 45% and 70% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR513W or its homologue, for example glutathione reductase or glutaredoxin superfamily protein, particularly have deoxyribonucleotide metabolism, cytoplasm, coerce reaction, detoxifcation and/or the electron transport energy relevant and preserve under the activity situation about being enhanced of active protein (for example shown in Table II the 5th or 7 row, the 238th row) with film, preferably, give each fine chemicals, preferred Q9 be increased in 31% and 73% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER156C or its homologue, for example under the activity situation about being enhanced of Jia Ding arabidopsis thaliana protein F2K15.180 superfamily protein (for example shown in Table II the 5th or 7 row, the 237th row), preferably, give each fine chemicals, preferably free Q9 be increased in 32% and 58% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER174C or its homologue, for example reactive gsh dependency oxydo-reductase of hydrogen peroxide and superoxide radical or Trx homology superfamily protein, for example have protein modification, coerce under the activity situation about being enhanced of protein (for example shown in Table II the 5th or 7 row, the 236th row) of reactive behavior, preferably, give each fine chemicals, preferred Q9 be increased in 31% and 72% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YPR172W or its homologue (shown in Table II the 5th or 7 row, the 235th row) is enhanced, preferably, give each fine chemicals, preferably free Q9 be increased in 34% and 66% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YBR184W or its homologue (shown in Table II the 5th or 7 row, the 232nd row) is enhanced, preferably, give each fine chemicals, preferably free Coenzyme Q10 99.0 be increased in 70% and 95% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YPR138C or its homologue, for example NH4 ⁺Transporter or ammonium transporter or ammonium transporter nrgA superfamily protein particularly have anion transport body (Cl, SO ⁴, PO ⁴Deng), other cation transfer body (Na, K, Ca, NH ⁴Deng), under the activity of the transhipment of nitrogen and sulphur, cell input and/or the active protein of plasma membrane (for example shown in Table II the 5th or 7 row, the 231st row) situation about being enhanced, preferably, give each fine chemicals, preferably free Coenzyme Q10 99.0 be increased in 65% and 257% or more between.

[0046.0.18.18] in one embodiment, as disclosed protein in [0016.0.18.18] or its homologue (shown in Table I the 5th or 7 row, 231-234 are capable), for example have such as in [0022.0.18.18] the active activity of proteins of definition be enhanced, preferably, giving each fine chemicals, preferred Coenzyme Q10 99.0 and other has VITAMIN or the active compound of coenzyme or its precursor and increases.

In one embodiment, as disclosed protein in [0016.0.18.18] or its homologue (shown in Table I the 5th or 7 row, 235-242 and/or 599-602 are capable), for example have as

The active activity of proteins that define is enhanced in [0022.0.18.18], preferably, gives each fine chemicals, preferably Coenzyme Q10 99.0 and other have VITAMIN or active compound of coenzyme or the increase of its precursor.

In one embodiment, one or more yeast saccharomyces cerevisiaes or Escherichia coli protein YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 or have as active homologue as described in top [0045.0.18.18], for example Table II the 5th or 7 is listed as, the activity of the homologue shown in 231-234 and/or 235-242 and/or 599-602 are capable is enhanced, and gives each fine chemicals, preferred each fine chemicals and/or comprise the Q9 of increase and/or the wax of Coenzyme Q10 99.0 level, film, membrane portions, organoid, triacylglycerol, lipid, oils and/or fat increase.

[0047.0.0.18] sees [0047.0.0.0]

[0048.0.18.18] each fine chemicals can free form and/or is contained in the biology with the form of the other parts of lipid, wax, film, membrane portions, protein, lipid acid or film (particularly plastosome or plastid film are comprised compound) bonded form or its mixture.Therefore, in one embodiment, film, organoid, cell, tissue, more preferably biological, quantity as free form in plant or microorganism or its part increases by 3% or higher, preferred especially 10% or higher, very particularly preferably be higher than 30% and most preferably 70% or higher, as 100%, 300% or 500%.Therefore, in another embodiment, film, organoid, cell, tissue, more preferably biological, quantity as each fine chemicals of bonded in plant or microorganism or its part increases by 3% or higher, preferred especially 10% or higher, very particularly preferably be higher than 30% and most preferably 70% or higher, as 100%, 300% or 500%.

[0049.0.18.18] has to give and improves each fine chemicals quantity or the active protein of level has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the sequence of consensus sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or as Table II the 5th row, its function homologue shown in 231-234 and/or 235-242 and/or 599-602 are capable described in polypeptide or the literary composition is (for example as Table II the 7th row, shown in 231-234 and/or 235-242 and/or 599-602 are capable), perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 231-234 and/or 235-242 and/or 599-602 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity described in the literary composition, particularly give Coenzyme Q10 99.0 and/or the increase of Q9 level level in the biology.

[0050.0.18.18]./.

[0051.0.18.18] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example hydrophobic or lipophilic composition.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of each fine chemicals and multiple other coenzyme, VITAMIN and/or antioxidant (for example vitamin B6 or vitamin-E).

[0052.0.0.18] sees [0052.0.0.0]

[0053.0.18.18] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention or the used polypeptide of the inventive method (for example have YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein, for example as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue are (for example as Table II the 7th row, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) express increase, have the raising Coenzyme Q10 99.0 described in the literary composition and/or the activity of Q9;

(b) stable mRNA, described mRNA gives nucleic acid molecule of the present invention or the coded protein of the inventive method nucleic acid molecule used therefor (for example has YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein, for example Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue are (for example as Table II the 7th row, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) express to improve or mRNA that coding has an active polypeptide of the present invention of raising Coenzyme Q10 99.0 described in the literary composition and/or Q9 expresses and improves;

(c) improve the protein specific activity, described protein is given the active of the raising Coenzyme Q10 99.0 that has described in the literary composition and/or Q9, (for example has YPR138C by the coded protein of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used polypeptide of the inventive method, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein, for example Table II the 5th is listed as, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention or the used polypeptide of the inventive method;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given the active of the raising Coenzyme Q10 99.0 that has described in the literary composition and/or Q9, by nucleic acid molecule of the present invention or the coded protein of the inventive method nucleic acid molecule used therefor, polypeptide perhaps of the present invention or the used polypeptide of the inventive method (for example have YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein, for example Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table I or II the 7th are listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) express and improve;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein is given the active of the raising Coenzyme Q10 99.0 that has described in the literary composition and/or Q9, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein are as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table I or II the 7th are listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) express and improve;

(f) transgenosis of expression coded protein, described protein is given the raising Coenzyme Q10 99.0 that has described in the literary composition and/or the activity of Q9, (for example has YPR138C by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein are as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table I or II the 7th are listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, described nucleic acid molecule encoding has the active of raising Coenzyme Q10 99.0 described in the literary composition and/or Q9, (for example has YPR138C by nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor encoded polypeptide or polypeptide of the present invention or the used polypeptide of the inventive method, YBR184W, b2699, b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, b0175 protein is as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table I or II the 7th are listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide);

(h) the negative Expression element of positive Expression element or removal improves code book invention polypeptide or the used polypeptide of the inventive method (for example has YPR138C by adding, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein, as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table I or II the 7th are listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or remove straining element from control region.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.18.18] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded polypeptide or having YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, (for example Table II the 5th is listed as the polypeptide of b0730 and/or b0175 protein active, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable) or its homologue (Table I or II the 7th row for example, polypeptide shown in 231-234 and/or 235-242 and/or 599-602 are capable) giving Coenzyme Q10 99.0 and/or Q9 after active expression of polypeptides or the activity increases.

[0055.0.0.18] to [0067.0.0.18] sees that [0055.0.0.0] is to [0067.0.0.0]

[0068.0.9.9] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of Q9 and/or Coenzyme Q10 99.0.

[0069.0.0.18] sees [0069.0.0.0]

[0070.0.18.18] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor or polypeptide of the present invention or the used expression of polypeptides of the inventive method, YPR138C perhaps encodes, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, (for example Table II the 5th is listed as for b0730 and/or b0175 protein, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable) or its homologue (Table II the 7th row for example, shown in 231-234 and/or 235-242 and/or 599-602 are capable) a gene or a plurality of gene import in biology separately or with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example contain high level (from the physiology of nutrition angle) coenzyme, the favourable hydrophobic composition of VITAMIN and/or antioxidant (for example vitamin B6 and/or vitamin-E).In one embodiment, protein shown in Table II the 5th row, 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, 231-234 and/or 235-242 and/or 599-602 capable shown in the expression of the protein of nucleic acid molecule encoding or its homologue (for example Table II the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in) give coenzyme, particularly Q9 and/or Coenzyme Q10 99.0 increases, and preferably give other coenzyme, VITAMIN and/or antioxidant and increase.

[0071.0.18.18] preferably, with composition of the present invention or composition biological or that its part provides also contain higher amount to the nutrition of animal and human's class or healthy have the metabolite that just influencing or low quantity to nutrition of animal and human's class or healthy metabolite with negatively influencing.Similarly, can improve and input or output nutrition or metabolite (comprises that cell biological synthesizes the required amino acid of each fine chemicals, lipid acid, VITAMIN, coenzyme, in antioxidant etc. or its precursor any one) number or the activity of other required genes are to improve organoid (for example plastosome or plastid), (the isoprenoid for example of necessity or associated precursors in cell or the corresponding storage compartment, acetyl-CoA, HMG-CoA, mevalonic acid, isopentenylpyrophosphate, geranylpyrophosphate, farnesylpyrophosphate) or the concentration of other cofactor or intermediate product.Because the raising or the active new generation of polypeptide of the present invention or the used polypeptide active of the inventive method, perhaps (for example (for example press down first hydroxyl enzyme element by the enzyme that improves synthetic precursor owing to the raising of nucleotide sequence of the present invention or the used nucleic acid sequence of the inventive method and/or to participating in biosynthetic other generegulation of each fine chemicals, the HMG-CoA reductase enzyme, Mevalonic kinase) active or by destroying the activity that participates in one or more genes that fine chemicals decomposes), can in host living beings such as plant or microorganism, improve the output of each fine chemicals, produce and/or produce efficient.

[0072.0.18.18] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds is antioxidant, other coenzyme, VITAMIN such as vitamin B6 or vitamin-E or comprises Coenzyme Q10 99.0 and/or the triacylglycerol of Q9, lipid acid, lipid, oils and/or fat.

[0073.0.18.18] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, organoid, plant or animal tissues or plant;

(b) improve YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, (for example Table II the 5th is listed as for b0730 and/or b0175 protein active or the coded polypeptide of nucleic acid molecule of the present invention, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable) (for example Table II the 7th is listed as active or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) activity, for example give biology (preferred microorganism, the non-human animal, plant or zooblast, plant or animal tissues or plant) in each fine chemicals improve;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis, and optional other free and/or bonded VITAMIN, coenzyme or antioxidant.

[0074.0.18.18] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues, organoid or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded or free and each fine chemicals of bonded, and if dissociate or/and bonded antioxidant, VITAMIN or coenzyme and composition thereof as selecting to produce, reclaim and expect to separate other.

[0075.0.0.18] to [0077.0.0.18] sees that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.18.18] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes, disclosing for example: 6 according to following U.S. Patent application, 380,252:Use of L-acetylcarnitine, L-isovalerylcarnitine, L-propionylcarnitine for increasingthe levels of IGF-1; 6,372,198:Dentifrice for the mineralization andremineralization of teeth; 6,368,617:Dietary supplement; 6,350,473:Method for treating hypercholesterolemia, hyperlipidemia, andatherosclerosis; 6,335,361:Method for the treatment of benign forgetfulness; 6,329,432:Mesozeaxanthin formulations for treatment of retinaldisorders; 6,328,987:Cosmetic skin care compositions containing alphainterferon; 6,312,703:Compressed lecithin preparations; 6,306,392:Composition comprising a carnitine and glutathione, useful to increase theabsorption of glutathione and synergize its effects; 6,303,586:Supportivetherapy for diabetes, hyperglycemia and hypoglycemia; 6,297,281:Association of no syntase inhibitors with trappers of oxygen reactiveforms; 6,294,697:Discrete-length polyethylene glycols; 6,277,842:Dietary supplemental method for fat and weight reduction; 6,261,250:Method and apparatus for enhancing cardiovascular activity and healththrough rhythmic limb elevation; 6,258,855:Method of retarding andameliorating carpal tunnel syndrome; 6,258,848:Methods andcompositions for increasing insulin sensitivity; 6,258,847:Use of 2-mercaptoethanolamine (2-MEA) and related aminothiol compounds andcopper (II)-3,5 di-isopropyl salicylates and related compounds in theprevention and treatment of various diseases; 6,255,354:Preparation of apulmonary surfactant for instillation and oral application; 6,254,547:Breath methylated alkane contour:a new marker of oxidative stress anddisease; 6,248,552:Enzyme-based assay for determining effects ofexogenous and endogenous factors on cellular energy production; 6,248,363:Solid earriers for improved delivery of active ingredients inpharmaceutical compositions; 6,245,800:Method of preventing ortreating statin-induced toxic effects using L-carnitine or an alkanoyl L-carnitine; 6,245,378:Nutritional supplement for facilitating skeletalmuscle adaptation to strenuous exercise and counteracting defatigation inasthenic individuals; 6,242,491:Use of creatine or creatine compounds forskin preservation; 6,232,346:Composition for improvement of cellularnutrition and mitochondrial energetics; 6,231,836:Folic acid dentifrice; 6,228,891:Use of 2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone; 6,228,402:Xylitol-containing non-human foodstuff andmethod; 6,228,347:Antioxidant gel for gingival conditions; 6,218,436:Pharmaceutically active carotenoids; 6,203,818:Nutritional supplementfor cardiovascular health; 6,200,550:Oral care compositions comprisingcoenzyme Q10; 6,191,172:Water-soluble compositions of bioactivelipophilic compounds; 6,184,255:Pharmaceutical compositioncomprising coenzyme Q10; 6,166,077:Use of L-acetylcarnitine, L-isovalerylcarnitine, L-propionylcarnitine for increasing the levels of IGF-1; 6,162,419:Stabilized ascorbyl compositions; 6,159,508:Xylitol-containing non-human foodstuff and method; 6,159,476:Herbalsupplement for increased muscle strength and endurance for athletes; 6,153,582:Defined serumfree medical solution for ophthalmology; 6,136,859:Pharmaceutical formulation for treating liver disorders; 6,107,281:Compounds and their combinations for the treatment ofinfluenza infection; 6,106,286:Method and device for administeringmedicine to the periodontium; 6,099,854:Dry composition containingflavonol useful as a food supplement; 6,086,910:Food supplements; 6,080,788:Composition for improvement of cellular nutrition andmitochondrial energetics; 6,069,167:Use of antioxidant agents to treatcholestatic liver disease; 6,063,820:Medical food for diabetics; 6,054,261:Coenzyme Q.sub.10 compositions for organ protection during perfusion; 6,051,250:Process for the stabilization of vesicles of amphiphilic lipid (s) and composition for topical application containing the said stabilizedvesicles.Can pass through highly basic hydrolysis, extraction and crystallization or by thin-layer chromatography with those skilled in the art are known and hereinafter described additive method, in a usual manner purified fermentation broth, tunning, plant or plant product.These are different checks that the product of steps is lipid acid or the fatty acid composition that still contains fermented liquid, vegetable particle and different quantities cellular component, and the favourable fermented liquid and the scope of cellular component are 0 to 99% by weight, preferably are lower than 80%, especially preferably are lower than 50%.

[0079.0.0.18] to [0084.0.0.18] sees that [0079.0.0.0] is to [0084.0.0.0]

[0084.2.18.18] it is reported, Agrobacterium (Agrobacterium sp.), protaminobacter ruber (Protaminobacter rubber) and Paracoccus denitrificans (Paracoccus denitrificans) produce Coenzyme Q10 99.0.It is reported that candida tropicalis (Candida tropicalis) produces Q9.It is reported, controllable continuous is cultivated the ubiquinone that phototrophic bacteria (wild type strain of the outer sulphur rhodospirillum (Ectothiorhodospira shaposhnikovii) of the red bacterium of pod membrane (Rhodobacter capsulatus), the red bacterium of class ball (Rhodobacter sphaeroides), pink pod sulphur bacterium (Thiocapsa roseopersicina) and Sha Shi) can produce the side chain lengths that has 6-10 unit, for example comprises Coenzyme Q10 99.0 and Q9.Cell mainly comprises a kind of main ubiquinone, so content and composition depend on growth conditions, substrate and other factor.Preferably in a kind of described biology or any other microorganism, produce greater than 0.1, be preferably greater than the 1-6mg/g stem cell, even preferred generation is greater than 10mg/g stem cell, 20mg/g stem cell, 50mg/g stem cell, 100mg/g stem cell, 200mg/g stem cell, 300mg/g stem cell, 500mg/g stem cell or more.

[0084.0.0.18] sees [0084.0.0.0]

[0085.0.18.18] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as nucleotide sequence or derivatives thereof shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.18] to [0087.0.0.18] sees that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.18.18] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified coenzyme content, particularly its Q9 and/or Coenzyme Q10 99.0 content.Because the nutritive value that for example is used for feed or nutraceutical plant depends on above-mentioned coenzyme, particularly essential Coenzyme Q10 99.0 and as the total number of the coenzyme of feed and food source, so this is very important for the plant breeder.Improving or producing YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein are (for example as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) after the activity, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, the transgenic plant that produced grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0088.1.0.18] sees [0088.1.0.0]

[0089.0.0.18] to [0094.0.0.18] sees that [0089.0.0.0] is to [0094.0.0.0]

[0095.0.18.18] advantageously, the pond that increases each fine chemicals by method of the present invention in genetically modified organism is to separate a large amount of pure basically fine chemicals.

[0096.0.18.18] is in another embodiment preferred of the present invention, improving nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide (for example coenzyme precursors (for example isoprenoid, acetyl-CoA, HMG-CoA, mevalonic acid, isoamyl tetra-sodium, geranylpyrophosphate, farnesylpyrophosphate etc.) transporter protein) or compound (improve coenzyme precursors produce) combines and can be used for producing each fine chemicals and (see Bao and Ohlrogge, Plant Physiol.1999 August; 120 (4): 1057-1062).

The content that [0097.0.18.18] increases each fine chemicals of contaminated with lipid also is favourable.

[0098.0.18.18] in preferred embodiments, each fine chemicals be produce according to the present invention and (if expectation) isolating.Produce other antioxidant, VITAMIN or coenzyme by method of the present invention, for example coenzyme Q-10, Q1, Q2, Q3, Q4, Q5, Q6, Q7 and/or Q8 or retinene, vitamin-E, vitamin B6 or other liposoluble vitamin or its mixture can be advantageously used in and produce the composition of using in food and/or feed or the cosmetics production.

For microbial fermentation, aforementioned coenzyme can be accumulated in membrane-bound fragment and/or lipotropy or the hydrophobic parts [0099.0.18.18].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate coenzyme or lipotropy or hydrophobic composition.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.18.18] comprises the transgenic plant of the method according to this invention synthetic Q9 and Coenzyme Q10 99.0 and can advantageously directly put on market, and the membrane-bound fragment, cell, cell fragment, organoid, plastid, wax, oils, lipid or the fat that do not need institute's synthetic to be comprised each fine chemicals separate.For example, because coenzyme can be from film (plastosome or plastid film) separation, so can only separate the cell part that comprises described film or described membrane-bound fragment.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.Yet, the fine chemicals that produces according to the inventive method can also from biological, advantageously from plant with the isolated in form of oils, fat, lipid and/or free fatty acids.Yet each fine chemicals that the method according to this invention produces can also be with the form of its cell, cell fragment, plastid, organoid, film, membrane-bound fragment, wax, oils, fat, lipid and/or lipid acid from biology, advantageously separate from plant.Can obtain the Q9 and the Coenzyme Q10 99.0 that produce by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part, preferred plant seed.In order to improve the efficient that oil extracts, with vegetable material, particularly seed clean, softening (temper) and to shell in case of necessity and peel off be favourable.In this article, oils, fat, lipid, wax and/or fatty acid part can need not heat cold be beaten or cold-press process obtains by being called.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.The composition that comprises the chemical pure fine chemicals can advantageously be applied to food or feed industry, cosmetic industry, particularly pharmacy industry.

[0101.0.18.18] sees [0101.0.0.0]

[0102.0.18.18] for example, coenzyme can advantageously detect by the LC separation method.By using standard method of analysis (as LC-MS, LC-MSMS or TLC) can clearly detect the existence of coenzyme product to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed; Also can be referring to Biotechnology of Vitamins, Pigments and Growth Factors, Erik J.Vandamme compiles, London, 1989, the 96-103 pages or leaves.

[0103.0.18.18] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as the sequence shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule is as shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding is opened by monoclonal antibody isolated polypeptide from expression library for example and is given the nucleic acid molecule that each fine chemicals quantity increases in biology or its part, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise as consensus sequence shown in Table IV the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0103.1.18.18] in one embodiment, the sequence difference was one or more Nucleotide shown in the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 were capable.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide of sequence.

[0103.2.18.18] in one embodiment, one or more sequence differences were one or more Nucleotide shown in the inventive method nucleic acid molecule used therefor and Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 were capable.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in one or more sequences form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIB the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide of sequence.

[0104.0.18.18] in one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 231-234 and/or 235-242 and/or the capable listed sequence difference of 599-602 are one or more Nucleotide, perhaps not by Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide of sequence.

[0105.0.0.18] to [0107.0.0.18] sees that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.18.18] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable, from Table II the 5th or 7 row, aminoacid sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable is derived or is listed as from containing Table IV the 7th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable, perhaps its coding has YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, its expression or active derivative or homologue are depended in proteinic enzymic activity of b0730 and/or b0175 or bioactive polypeptide or Coenzyme Q10 99.0 and Q9 increase.

[0109.0.0.18] sees [0109.0.0.0]

[0110.0.18.18] helps the inventive method and encodes to have the polypeptide of polypeptide of the present invention or the used polypeptide active of the used or of the present invention process of the inventive method, for example YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein are (for example as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by as Table I the 5th row, the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue are (as Table II the 7th row, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) nucleic acid molecule can from generally open database, determine.

[0111.0.0.18] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.18.18] the inventive method is the isolated nucleic acid sequences form, its coding has YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein active are also given Coenzyme Q10 99.0 and/or the polypeptide of Q9 increase, for example as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table I or II the 7th are listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable).

[0113.0.0.18] to [0117.0.0.18] sees that [0113.0.0.0] is to [0117.0.0.0]

[0118.1.0.18] in one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor are from the plant with high Coenzyme Q10 99.0 and/or Q9 content.In one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor from and/or transformed and to be entered plant with high Coenzyme Q10 99.0 and/or Q9 content.

[0118.0.0.18] to [0120.0.0.18] sees that [0118.0.0.0] is to [0120.0.0.0]

[0121.0.18.18] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, peptide sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or its functional homologue as herein described have the difference of one or more amino acid moleculars, described artificial sequence is preferably given aforementioned activity, promptly improving its activity, for example YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, (for example Table II the 5th is listed as for b0730 and/or b0175 protein, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) give each fine chemicals after the activity and increase.

[0122.0.0.18] to [0127.0.0.18] sees that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.18.18] polymerase chain reaction (PCR) amplification is used (for example as the primer shown in Table III the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence or from producing as sequence deutero-sequence shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.

[0129.0.18.18] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention or the used polypeptide of the inventive method is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence was from described comparison shown in Table IV the 7th row, 231-234 and/or 235-242 and/or 599-602 were capable.

[0130.0.18.18] can use degenerated primer to have aforementioned active new protein fragment by pcr amplification then, and described activity is for example improving YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein active or from other functional homologue of other biological polypeptide of the present invention (Table II the 5th row for example, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) expression or activity after give each fine chemicals, particularly Coenzyme Q10 99.0 and/or Q9 increase.

[0131.0.0.18] to [0138.0.0.18] sees that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.18.18] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly give each fine chemicals level increase), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence hybridization.

[0140.0.0.18] to [0146.0.0.18] sees that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.18.18] in addition, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise one of the nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it with Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in one of nucleotide sequence fully complementary, thereby one of nucleotide sequence shown in can be with Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable is hybridized and is formed stable duplex.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.18.18] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor contain following nucleotide sequence: with Table I the 5th or 7 row, nucleotide sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or its functional portion homologous are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly improving YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or the b0175 gene product (Table II of for example encoding the 5th row, proteinic gene shown in 231-234 and/or 235-242 and/or 599-602 are capable) active or comprise or expresses and be listed as Table I the 5th, (for example Table I or II the 7th are listed as for nucleic acid molecule shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) increase each fine chemicals afterwards, particularly Coenzyme Q10 99.0 and/or Q9.

[0149.0.18.18] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise nucleotide sequence, described sequence and Table I the 5th or 7 row, one of nucleotide sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (as giving the fine chemicals increase) and randomly has YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, (for example Table II the 5th is listed as for b0730 and/or b0175 protein, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue (Table I for example or II the 7th row, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) active protein.

[00149.1.18.18] randomly, with Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity is for being known activity or being used for these protein of note as the protein shown in Table II the 3rd row, 231-234 and/or 235-242 and/or 599-602 are capable.

[0150.0.18.18] in addition, nucleic acid molecule of the present invention or process nucleic acid molecule used therefor of the present invention can only contain Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table I B the 7th row, the part of the coding region of one of nucleotide sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give Coenzyme Q10 99.0 and/or Q9 increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and described nucleotides sequence column region is listed as with Table I the 5th or 7 under stringent condition, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table I B the 7th row, the sense strand of one of sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable, Table I the 5th or 7 row, the antisense sequences of one of sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or its be natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotide hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, the right PCR of primer shown in 231-234 and/or 235-242 and/or 599-602 are capable will produce YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, (for example coding is as Table II the 5th row for b0730 and/or b0175 gene product, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table II B the 7th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or expression are as Table I the 5th row, (for example Table II the 7th is listed as for nucleic acid molecule shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) gene) fragment.

[0151.0.0.18] sees [0151.0.0.0]

[0152.0.18.18] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coded polypeptide or its part, described polypeptide or its part comprise and are listed as Table II the 5th or 7,231-234 and/or 235-242 and/or 599-602 are capable, preferred Table I B the 7th row, the abundant homology of aminoacid sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or each fine chemicals of raising as be shown in the examples, the activity that produces of Coenzyme Q10 99.0 and/or Q9 particularly.

[0153.0.18.18] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as protein shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable or its part have Table II the 3rd row for example as herein described, 231-234 and/or 235-242 and/or 599-602 capable shown in the activity of polypeptide.

[0154.0.18.18] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.18] to [0156.0.0.18] see[0155.0.0.0] to [0156.0.0.0]

[0157.0.18.18] the present invention relates in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, one of nucleotide sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable (with its part) and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 7th row, the polypeptide of consensus sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or as Table II the 5th or 7 row, the nucleic acid molecule of polypeptide shown in 231-234 and/or 235-242 and/or 599-602 are capable or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as the aminoacid sequence shown in Table IV the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable or as the aminoacid sequence of polypeptide shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable or its function homologue.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, aminoacid sequence or the basic homology of function homologue shown in described full length protein and Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.Yet, in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or the 599-602 sequence shown in capable.Preferably, nucleic acid molecule of the present invention is a functional homologous compound or identical with nucleic acid molecule shown in Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable.

[0158.0.0.18] to [0160.0.0.18] sees that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.18.18] therefore, in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor length are at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.18] sees [0162.0.0.0]

[0163.0.18.18] preferably, under stringent condition with Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in the nucleic acid molecule of the present invention of sequence hybridization or the inventive method nucleic acid molecule used therefor corresponding to naturally occurring nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.18] sees [0164.0.0.0]

[0165.0.18.18] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.18] to [0167.0.0.18] sees that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.18.18] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table II B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table II B the 7th row, 231-234 and/or 235-242 and/or the 599-602 aminoacid sequence shown in capable is at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table II B the 7th row, sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table II B the 7th row, one of sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table II B the 7th row, sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table II B the 7th row, sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.18] to [0172.0.0.18] sees that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.18.18] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:22609 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:22609 sequence before use.

[0174.0.0.18] sees [0174.0.0.0]

[0175.0.18.18] for example, the sequence that has 80% homology at protein level and SEQ ID NO:22610 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:22610 sequence.

[0176.0.18.18] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtained shown in 231-234 and/or 235-242 and/or 599-602 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 231-234 and/or 235-242 and/or 599-602 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in 231-234 and/or 235-242 and/or 599-602 are capable has essentially identical character and discerns.

[0177.0.18.18] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the functional equivalent that nucleotide sequence obtained shown in 231-234 and/or 235-242 and/or 599-602 were capable be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide had at least 30% shown in 231-234 and/or 235-242 and/or 599-602 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in 231-234 and/or 235-242 and/or 599-602 are capable.

[0178.0.0.18] sees [0178.0.0.0]

[0179.0.18.18] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table I B the 7th row, shown in 231-234 and/or 235-242 and/or 599-602 are capable) the middle replacement of introducing one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, 231-234 and/or 235-242 and/or 599-602 are capable, preferred Table II B the 7th row, the nucleic acid molecule of the homologue of protein sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 introduce sudden change in the encoding sequence of sequence shown in capable.

[0180.0.0.18] to [0183.0.0.18] sees that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.18.18] is employed to have as Table I the 5th or 7 row, the homologous compound of the nucleotide sequence of sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.18.18] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or the 599-602 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, other Nucleotide of not showing in preferred Table I B the 7th row, 231-234 and/or 235-242 and/or the 599-602 arbitrary sequence shown in capable.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or the 599-602 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.18.18] also preferred the inventive method comprise as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table II B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table II B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence identical.

[0187.0.18.18] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table II B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in encoding sequence identical.

The polypeptide (=protein) that [0188.0.18.18] still has the basic the enzyme activity of polypeptide of the present invention (being its active basic reduction that do not have) of giving each fine chemicals and increasing is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, actively do not reduce substantially with comparing as polypeptide expressed activity shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable and under the same conditions.In one embodiment, polypeptide of the present invention is to comprise as sequence shown in Table II B the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable or by its homologue of forming.

[0189.0.18.18] as the homologous compound of nucleotide sequence shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.18] to [0191.0.0.18] sees that [0190.0.0.0] is to [0191.0.0.0]

[0191.1.18.18] in one embodiment, compare with biological or its part of described contrast or selection, according to the inventive method described herein should biology or its part provide improve the standard in conjunction with any wax, triacylglycerol, lipid oils and/or fat fine chemicals or comprise any combination or any composition of free Coenzyme Q10 99.0 and/or Q9, for example be bonded to lipid, lipoprotein, membrane portions, sphingolipid, phosphoglyceride, lipid, glycolipid class such as sphingoglycolipid, phosphatide such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, the glycerine Serine, phosphatidylinositols or two glycerine Serine or monoacylglycerols, diacylglycerol or triacylglycerol.

[0192.0.0.18] to [0203.0.0.18] sees that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.18.18] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table II B the 7th row, 231-234 and/or 235-242 and/or 599-602 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given each fine chemicals quantity in biological or its part increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or 599-602 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as the primer shown in Table III the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable or primer amplification from cDNA library or genomic library is obtained by using, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 231-234 and/or 235-242 and/or 599-602 capable shown in consensus sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(k) coded polypeptide aminoacid sequence and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described peptide coding is as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, the structural domain of preferred Table II B the 7th row, 231-234 and/or 235-242 and/or the 599-602 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, nucleic acid molecule shown in 231-234 and/or 235-242 and/or 599-602 are capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 231-234 and/or 235-242 and/or 599-602 are capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence form.In another embodiment, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In other embodiments, nucleic acid molecule not coding schedule IIA the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide of sequence.Therefore, in one embodiment, polypeptide had the different polypeptide of one or more amino acid at least shown in nucleic acid molecule encoding of the present invention and Table II A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 were capable, but coding schedule IIA the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in the protein of sequence.Therefore, in one embodiment, by nucleotide sequence (a)-(I) encoded protein matter not by Table II A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in protein sequence at least 30% identical and with Table II A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% identical.

Thereby, in another embodiment, (a) be different from as the sequence shown in Table I B the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable by one or more Nucleotide to the nucleic acid molecule of (l).In one embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor not by Table I B the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence form.In another embodiment, nucleic acid molecule of the present invention and Table I B the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In other embodiments, nucleic acid molecule not coding schedule IIB the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide of sequence.Therefore, in one embodiment, polypeptide had the different polypeptide of one or more amino acid at least shown in nucleic acid molecule encoding of the present invention and Table II B the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 were capable, but coding schedule IIB the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in the protein of sequence.Therefore, in one embodiment, by nucleotide sequence (a)-(I) encoded protein matter not by Table II B the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence form.In another embodiment, protein of the present invention and Table II B the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in protein sequence at least 30% identical and with Table II A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence less than 100%, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% identical.

[0205.0.0.18] to [0226.0.0.18] sees that [0205.0.0.0] is to [0226.0.0.0]

[0227.0.18.18] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table I B the 7th row, 231-234 and/or 235-242 and/or the capable sequence or derivatives thereof of mentioning of 599-602, can additionally advantageously express and/or other genes that suddenly change in biology.Particularly advantageously, extra other genes of expressing at least one isoprenoid biosynthetic pathway in biological (as plant or microorganism), for example for example HMG-CoA reductase enzyme, Mevalonic kinase etc. of other gene of acetyl-CoA, HMG-CoA, mevalonic acid, isoamyl tetra-sodium, geranylpyrophosphate, farnesylpyrophosphate biosynthetic pathway.Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required isoprenoid, coenzyme precursors or coenzyme, preferred Q9 and/or Q10, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0227.1.18.18] except Table I the 5th or 7 row, 231-234 and/or 235-242 and/or the capable sequence or derivatives thereof of mentioning of 599-602, can also advantageously knock out and/or mutation biology in other gene.For example, if the one or more genes in isoprenoid or the ubiquinone pathways metabolism are lowered, delete or knocked out in another way in such as the biology of plant or microorganism, then be favourable.

[0228.0.18.18] therefore cultivated to cross simultaneously in another embodiment of the present invention and expressed coding and isoprenoid metabolism (particularly Q9 or Coenzyme Q10 99.0 are synthetic) at least one nucleic acid of related protein or the biology of a gene.

[0229.0.18.18]./.

[0230.1.0.18]./.

[0230.2.0.18]./.

[0230.0.0.18] sees [0230.0.0.0]

[0231.0.18.18] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded Coenzyme Q10 99.0 and/or Q9 simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.18] to [0276.0.0.18] sees that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.18.18] can separate the coenzyme that produces by the method that the technician is familiar with from biology, for example precipitate by above-mentioned extraction, salt and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.Each fine chemicals of producing by this process can as above-mentioned from biological (advantageously being plant) isolated in form with its wax, membrane portions, oils, fat, lipid and/or lipid acid.The fraction that comprises coenzyme of producing by this process can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing or extract realization with plant part, preferred plant seed.

[0278.0.0.18] to [0283.0.0.18] sees that [0278.0.0.0] is to [0283.0.0.0]

[0283.0.18.18] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly be anti-YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein antibody or anti-as Table II the 5th or 7 row, the antibody of polypeptide or its antigen part shown in 231-234 and/or 235-242 and/or 599-602 are capable, it can utilize polypeptide of the present invention or its fragment of polypeptide that the inventive method characterizes (being polypeptide of the present invention) to produce by standard technique.Preferably specificity is in conjunction with the monoclonal antibody as polypeptide shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.

[0284.0.0.18] sees [0284.0.0.0]

[0285.0.18.18] the present invention relates to have as sequence shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable in one embodiment.

[0286.0.18.18] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to and comprising more than a polypeptide as consensus sequence (each row) shown in Table IV the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable.

[0287.0.0.18] to [0289.0.0.18] sees that [0287.0.0.0] is to [0289.0.0.0]

[0290.0.0.18] sees [0290.0.0.0]

[0291.0.18.18] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention or the used polypeptide of the inventive method are different from as the sequence shown in Table II A or IIB the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.

[0292.0.0.18] sees [0292.0.0.0]

[0293.0.18.18] in one embodiment, the present invention relates in biological or its part, give that each fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention, and have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.In another embodiment, described polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide can't help Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.18.18] in one embodiment, the present invention relates to have YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, the polypeptide of b0730 and/or b0175 protein active, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable.

[0295.0.0.18] to [0296.0.0.18] sees that [0295.0.0.0] is to [0296.0.0.0]

The statement that [0297.0.18.18] " do not contain cellular material " substantially comprises that protein wherein produces the preparation of isolating polypeptide of the present invention its cellular component of cell or the used polypeptide of the inventive method from natural or reorganization.In one embodiment, the statement that " does not contain cellular material substantially " comprises and contains the preparation that is lower than about 30% (dry weight) " contaminating protein matter ", " the contaminating protein matter " more preferably less than about 20%, also more preferably less than about 10% " contaminating protein matter ", and most preferably be lower than the preparation of about 5% " contaminating protein matter ".Term " contaminating protein matter " relates to the polypeptide that is not polypeptide of the present invention.When reorganization produces polypeptide of the present invention or its biologically-active moiety, also preferably do not contain substratum substantially, promptly substratum less than the protein formulation cumulative volume 20%, be more preferably less than 10%, and most preferably less than 5%.The statement that " does not contain precursor or other chemical substantially " comprises polypeptide wherein of the present invention isolating preparation from the precursor that participates in protein synthesis or other chemical.The statement that " does not contain precursor or other chemical substantially " comprises and contains the precursor that is lower than 30% (dry weight) (Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) or non-YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, the chemical of b0730 and/or b0175, preferably be lower than 20% precursor (Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) or non-YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b3605, b2699, the chemical of b0730 and/or b0175, also more preferably less than 10% precursor (Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) or non-YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, the chemical of b0730 and/or b0175, and most preferably be lower than 5% precursor (Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) or non-YPR138C, YBR184W, b2699, b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, the preparation of the chemical of b0175.In preferred embodiments, isolating protein or its biologically-active moiety do not derive from the contaminating protein matter of the same biology that produces polypeptide of the present invention.Generally produce this proteinoid by recombinant technology.

[0298.0.18.18] polypeptide of the present invention or the used polypeptide of the inventive method can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602.

[0299.0.18.18] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of aminoacid sequence sequence shown in Table II A or IIB the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable.

[0300.0.18.18] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7,231-234 and/or 235-242 and/or 599-602 capable shown in sequence different.Therefore, this polypeptide contains and is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as complete amino acid sequence homology shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.18] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.18.18] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence of the aminoacid sequence shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.18] sees [0303.0.0.0]

[0304.0.18.18] operation nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor may cause produce different with wild-type protein, as the protein of nucleic acid molecule encoding shown in capable shown in Table II the 5th row, 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, 231-234 and/or 235-242 and/or 599-602 or its homologue (as Table II the 7th be listed as, 231-234 and/or 235-242 and/or 599-602 capable shown in).These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.18.18] causes that polypeptide of the present invention or polypeptide that the inventive method is used improve described active any mutagenesis strategy and all be meant nonrestrictively, and the variation of these strategies is that those skilled in the art are conspicuous.Use these the strategy and in conjunction with mechanism disclosed herein, can utilize nucleic acid molecule of the present invention and polypeptide or the inventive method nucleic acid molecule used therefor and polypeptide to produce plant or its part, described plant or its part are expressed encoding wild type YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, (for example Table II the 5th is listed as for b0730 and/or b0175 protein, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable)) or Table II the 5th or 7 row, shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein that nucleic acid molecule shown in 231-234 and/or 235-242 and/or 599-602 are capable is coded or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) mutant, Tu Bian YPR138C for example, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, proteinic nucleic acid molecule of the present invention of b0730 and/or b0175 and peptide molecule of the present invention or the used peptide molecule of the inventive method are to improve the yield of required compound, output and/or generation efficient.

[0306.0.0.18] to [0308.0.0.18] sees that [0306.0.0.0] is to [0308.0.0.0]

[0309.0.18.18] in one embodiment, " YPR138C; YBR184W; b2699 and/or b1829 and/or YPR172W; YER174C; YER156C; YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein (=polypeptide) " be meant the polypeptide that has corresponding polypeptide of the present invention or be used for the amino acid sequence of polypeptide of the inventive method; and " non-YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 polypeptide " or " other polypeptide " be meant the polypeptide of aminoacid sequence with corresponding following proteins; described protein and polypeptide of the present invention be homology not basically; preferably with have YPR138C; YBR184W; b2699 and/or b1829 and/or YPR172W; YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, the polypeptide of b0730 and/or b0175 protein active is homology not basically, does not for example give described in the literary composition active and from the protein of identical or different biology.In one embodiment, do not give biology or each fine chemicals increase of its part at " the non-polypeptide of the present invention " or " other polypeptide " of Table II the 5th or 7 row, 231-234 and/or 235-242 and/or the capable demonstration of 599-602.

[0310.0.0.18] to [0317.0.0.18] sees that [0310.0.0.0] is to [0317.0.0.0]

[0318.0.18.18] in an especially preferred embodiment, polypeptide of the present invention do not have in addition by Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in those coded proteinic sequences of sequence.

[0319.0.0.18] to [0334.0.0.18] sees that [0319.0.0.0] is to [0334.0.0.0]

[0335.0.18.18] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of sequence shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.18] to [0342.0.0.18] sees that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.18.18] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown between the genetic transcription thing of the nucleotide sequence of one of sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as one of sequence shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable or its homologue in a kind of organism.

[0344.0.0.18] to [0361.0.0.18] sees that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.18.18] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention or the inventive method nucleic acid molecule used therefor, nucleic acid construct of the present invention, antisense molecule of the present invention, (for example coding has YPR138C for carrier of the present invention or code book invention polypeptide, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein active or Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active polypeptide) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, cell activity improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Has YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 protein active, for example Table II the 5th is listed as, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) transgenosis of active polypeptide is meant in the text because genomic regulation and control or manipulation, YPR138C in cell or biological or its part, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 or YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175-sample activity are enhanced, for example Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) activity improve.Example and the inventive method are described in above.

[0363.0.0.18] sees [0363.0.0.0]

[0364.0.18.18] is when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---for example naturally occurring YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730 and/or b0175 bak promoter and corresponding YPR138C, YBR184W, b2699 and/or b1829 and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, the combination of b0730 and/or b0175 protein gene, for example Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) promotor and the combination of corresponding gene---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.18] to [0376.0.0.18] sees that [0365.0.0.0] is to [0376.0.0.0]

[0377.0.18.18] therefore, the invention still further relates to the method according to this invention, thus the coenzyme that is produced is separated to high purity, be preferably greater than 70%w/W or higher, more preferably 80% or higher, preferred especially 90%w/W or higher, even more preferably 95%w/W or higher, for example 97%w/W or higher, for example 98%w/w, 99%w/w or 99.9%w/w.

[0378.0.18.18] whereby can recovery, purifying or separate and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than 90% the Q9 or the Coenzyme Q10 99.0 that produce in the method.As required, resultant coenzyme (composition that for example comprises coenzyme) can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.18.18] in one embodiment, described coenzyme is each fine chemicals, preferred reductive Q9 and/or Coenzyme Q10 99.0.

The suitable synthetic parent material of the part that comprises Q9 or Coenzyme Q10 99.0 that [0380.0.18.18] obtains in the method for the invention or each fine chemicals as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises Q9 that separation produces or Coenzyme Q10 99.0 composition or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is each fine chemicals or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine that the inventive method produces.

[0381.0.0.18] to [0382.0.0.18] sees that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.5.18] is in order to prepare the fine chemicals that contains antioxidant, coenzyme and/or VITAMIN, each fine chemicals particularly can use organic compound such as wax, oils, fat and/or lipid or comprises the part of film, particularly comprises the composition of plastosome or plastid film as the source.

[0384.0.0.18] sees [0384.0.0.0]

[0385.0.18.18] with this kind approach for example particularly containing of obtaining fermented liquid with other lipid, fat and/or oils and/or other VITAMIN or coenzyme or antioxidant blended Coenzyme Q10 99.0 and/or Q9, dry matter content accounts for 1 to 50% of weight usually.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant) or these methods or partly from fermented liquid, shift out or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.18.18] yet, can also be further purified each fine chemicals that comprises of generation, for example the part of Q9 and/or Coenzyme Q10 99.0 or composition.For this reason, the composition that will contain product carries out thin-layer chromatography or carries out Florisil column chromatography (Bouhours J.F., J.Chromatrogr.1979,169,462) on silica-gel plate, and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.An alternative approach of purification of fatty acid is for for example carrying out crystallization under the urea existence condition.These methods can combination with one another.

[0387.0.0.18] to [0392.0.0.18] sees that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.18.18] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify the nucleic acid molecule of particularly under lax stringent condition, hybridizing, and randomly separate full length cDNA clone or complete genome group clone as the nucleic acid molecule shown in Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable with nucleic acid molecule of the present invention;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.18] to [0399.0.0.18] sees that [0394.0.0.0] is to [0399.0.0.0]

Whether [0399.1.18.18] it is contemplated that by for example to the resistance of blocking-up Coenzyme Q10 99.0 and/or Q9 synthetic medicine and observe this effect and depend on as Table II the 5th row, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable), each fine chemicals that screens raising produces, for example by comparing and having low and high being listed as Table II the 5th, protein shown in 231-234 and/or 235-242 and/or 599-602 are capable or by Table I the 5th row, (for example Table II the 7th is listed as for the protein of nucleic acid molecule encoding shown in 231-234 and/or 235-242 and/or 599-602 are capable or its homologue, shown in 231-234 and/or 235-242 and/or 599-602 are capable) active biological phenotype more much at one.

[0400.0.0.18] to [0416.0.0.18] sees that [0400.0.0.0] is to [0416.0.0.0]

[0417.0.18.18] nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor to isoprenoid, particularly Q9 and/or Coenzyme Q10 99.0 biosynthetic pathway has resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block each fine chemicals synthetic inhibitor in the described biology.

Because the damage that dihydrocoenzyme Q9 and/or Coenzyme Q10 99.0 can protect biological antioxidant to coerce, the level that therefore increases each fine chemicals can the anti-weedicide that causes producing oxygenated compound (for example singlet oxygen) of protective plant.Known in addition, but Q9 and/or Coenzyme Q10 99.0 stabilizing membrane.

Therefore, in one embodiment, the increase of each fine chemicals level is used for the anti-weedicide that destroys film or cell (because generation of free oxygen free radical) of protective plant.

Producing the inhibitor of oxidative stress or the example of weedicide is aryl triazoles (aryl triazion), for example sulfentrazone, fluorine ketazolam grass; Perhaps diphenyl ether, for example acifluorfen, lactofen or Oxyfluorfen; Perhaps N-phenylphthalimide, for example imide phenoxy acetic acid Huo Fu oxazinone; Substituted urea class, for example fluometuron, Metribuzin, Diuron Tech or methoxydiuron; Triazines, for example G-30027, prometryn, ametryn, sencorex, prometon, simazine or six piperazines are together; Perhaps uracil, for example bromacil or terbacil.

The inhibitor of isoprenoid approach also causes the toxic reduction of Q9 in cell or the film and/or Coenzyme Q10 99.0, for example Guangmieling.Therefore, in one embodiment, each fine chemicals that the present invention relates to improve according to the present invention is used for the purposes of the anti-carotenoid inhibitor of protective plant such as pyridine and pyridazinone.

[0418.0.0.18] to [0423.0.0.18] sees that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.18.18] therefore, nucleic acid of the present invention or the inventive method nucleic acid molecule used therefor, the used polypeptide of polypeptide of the present invention or the inventive method, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, agonist with the inventive method evaluation, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other antioxidants, coenzyme or VITAMIN, particularly vitamin-E, or vitamin B6.

Therefore, nucleic acid of the present invention or the inventive method nucleic acid molecule used therefor or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.18] sees [0425.0.0.0]

[0425.1.18.18] be two more than ten years in the past, there are many research report Coenzyme Q10 99.0s in the congestive heart failure treatment, to be used as the resulting positive findings of auxiliary, for example the antioxygenation of ATP, stabilizing cell membrane or reduction thrombocyte size and restriction biologically active pdgf in the Support Line plastochondria.Be used for the congestive heart failure treatment in Japan from Coenzyme Q10 99.0 in 1974 as the approval medicine.The early stage research of congestive heart failure concentrates on the essential DCM (dilated cardiomyopathy), uses the effect of the anti-placebo of echocardiography assess cardiac function test Coenzyme Q10 99.0.The echocardiographic improvement of being seen in these researchs normally slowly continues, and is accompanied by alleviating of fatigue, pectoralgia, expiratory dyspnea and palpitaition.In some patients, only use Coenzyme Q10 99.0 just to recover the size and the function of normal heart; This mainly betides among the patient who just sends out congestive heart failure recently.

Present known HMG-CoA reductase inhibitor also significantly reduces the level of Coenzyme Q10 99.0 in effective reducing cholesterol level.This may be extrahazardous for the patient who suffers from cardiac failure, and this may hint if not all also being Coenzyme Q10 99.0 level reduction in the individuality of these reducing cholesterol medicines of many uses.Have been noted that in ill gum significant Coenzyme Q10 99.0 lacks.Suppose that it is irrational that Coenzyme Q10 99.0 can help muscular dystrophy, cancer (for example reduce the tumour size, alleviate metastatic breast cancer), AIDS, neurodegenerative disorders (for example Huntington's disease, familial Alzheimer ' s disease, Parkinson's disease), male sterility (Coenzyme Q10 99.0 improves the sick protection of motility of sperm seminal fluid and avoids radical damage), chronic stable angina pectoris, the significantly reduction of plasma adiponectin levels of peroxide, skin photoage, other immune dysfunction.Muscular dystrophy is relevant with heart trouble usually.Some evidences show that Coenzyme Q10 99.0 can increase energy and quicken the recovery that the muscle relevant with exercise is tired out and damage in addition.

Therefore; the nucleic acid that the present invention or the inventive method are used; the polypeptide that the present invention or the inventive method are used; nucleic acid construct of the present invention; biology of the present invention; host cell; microorganism; plant; plant tissue; vegetable cell or its part; carrier of the present invention; the antagonist or the agonist that use the inventive method to identify; antibody of the present invention; antisense molecule of the present invention or the nucleic acid molecule that uses the inventive method to identify can be used in the preparation of medicine; for example comprise the fine chemicals that separates or reclaim; vegetables; lipid; fat or oils; particularly for example with other antioxidant; VITAMIN or coenzyme combination; be used for the treatment of congestive heart failure; myocardial dysfunction (for example being used for stabilizing cell membrane); the thrombocyte size of dwindling; limited biologically active pdgf; the essential DCM (dilated cardiomyopathy); tired; pectoralgia; expiratory dyspnea and palpitaition; other performance of hypertension and cardiovascular disorder; because the Coenzyme Q10 99.0 level of using the HMG-CoA reductase inhibitor to reduce; ill gum; gingivitis; cancer; AIDS; other immune dysfunction; the muscular dystrophy relevant with heart trouble; tired out and the damage of the muscle relevant with exercise; fat; neurodegenerative disorders (Huntington's disease for example; familial Alzheimer ' s disease (for example with the vitamin B6 combination); Parkinson's disease); male sterility; chronic stable angina pectoris; the plasma adiponectin levels of peroxide significantly reduces; skin photoage; the fat peroxidation perhaps is used to dwindle the tumour size; be used to alleviate metastatic breast cancer; be used to improve sperm motility; be used to be protected from oxidative stress or protection seminal fluid.Pharmaceutical composition may comprise other coenzyme VITAMIN, antioxidant or nutrition.As antioxidant, each fine chemicals is used for advantageous embodiment with the reduction form.

[0425.2.18.18] in addition, the nucleic acid that the present invention or the inventive method are used, the polypeptide that the present invention or the inventive method are used, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the antagonist or the agonist that use the inventive method to identify, antibody of the present invention, it is for example combined with pharmaceutically acceptable carrier that antisense molecule of the present invention or the nucleic acid molecule that uses the inventive method to identify can be used in pharmaceutical compositions, make-up composition for example with emulsifying agent, washing agent, dye additive, tenderizer, forming agent, wetting agent, lubricant or the like is combined, perhaps as nutritional additive for example as based on the capsule of oil, be full of capsule or the tablet or the solubility soft capsule (microemulsion or other) of powder.

Dosage range every day of Coenzyme Q10 99.0 is the 5-300 milligram.Use those people of Coenzyme Q10 99.0 treatment alveolysis to take in the 50-200 milligram every day, for example every day the 100-150 milligram.The same dose range applications is in those people that take statin pharmacological agent hypercholesterolemia.Coenzyme Q10 99.0 is preferably taken in food, particularly with fat or oils.There are about three weeks of report dosed administration every day the time must obtain the maximum serum-concentration of Coenzyme Q10 99.0.Coenzyme Q10 99.0 also can be attained at some toothpaste and skin cream in the part.

In a further embodiment, the present invention or the used nucleic acid of the inventive method that are used for pharmaceutical compositions, the polypeptide that the present invention or the inventive method are used, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the antagonist or the agonist that use the inventive method to identify, antibody of the present invention, antisense molecule of the present invention or the nucleic acid molecule that uses the inventive method to identify comprise each fine chemicals isolating or that reclaim, perhaps be contained in described biology of the present invention, host cell, microorganism, plant, plant tissue, the lipid of vegetable cell or its part, fat, in oils or the film component, for example further comprise other antioxidant, VITAMIN, nutritional additive or coenzyme be selenium for example, vitamin-E, vitamin B6, Vogan-Neu.

Can suppose Q9 can be fine be used for such use.Q9 only " afterbody " is different with Coenzyme Q10 99.0, and it is a shorter isoprenoid unit.Therefore, can suppose that Q9 has more weak lipotropy and stronger wetting ability than Coenzyme Q10 99.0.Therefore, in one embodiment, advantageously use the product or the product of the present invention of the inventive method to produce specific hydrophilization cosmetic compositions, for example combined with emulsifying agent, washing agent, dye additive, tenderizer, forming agent, wetting agent, lubricant or the like, perhaps produce the particular hydrophilic nutritional additive, for example based on the capsule of oil, the capsule that is full of powder or tablet or solubility soft capsule (microemulsion or other).

[0425.3.18.18] the present invention relates to the purposes that antagonist of the present invention, plant of the present invention or its part, microorganism of the present invention or host cell or polypeptide of the present invention or its part are used for production cosmetic compositions or pharmaceutical composition in other embodiments.This composition has anti-oxidant activity, light protection activity and/or tanning activity, but for example it is used for the treatment of high-level cholesterol and/or fat with pharmaceutically acceptable carrier or cosmetic carrier combinations; Be used for protection, treat or cure above-mentioned disease; Perhaps be used for cleaning, conditioning and/or handle skin.

Q9 and/or Coenzyme Q10 99.0 can be used as the stablizer of other color sensitivity or oxygen sensitive compound.

[0426.0.0.18] sees [0426.0.0.0]

[0426.1.0.18]./.

[0427.0.0.18] to [0430.0.0.0] sees that [0427.0.0.0] is to [0430.0.0.0]

[0431.0.18.18] embodiment 1: clone SEQ ID NO:22609 in intestinal bacteria

[0432.0.18.18] uses the method for known good foundation (to consult as Sambrook, J. etc. (1989) " Molecular Cloning:A Laboratory Manual " Cold Spring HarborLaboratory Press or Ausubel, F.M. etc. (1994) " Current Protocols in Mole-cular Biology ", John Wiley ﹠amp; Sons), the DNA polynucleotide that will have shown in Table I the 5th row sequence and coding as the listed polypeptide of following table 1, for example SEQ ID NO:22609 be cloned into plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl Acad.Sci.USA, 75:3737-3741); PACYC177 (Change ﹠amp; Cohen (1978) J.Bacteriol.134:1141-1156); PBS series plasmid (pBSSK+, pBSSK-and other; Stratagene, LaJolla, USA) or clay such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J.Rosenthal, A., and Waterson, R.H. (1987) Gene 53:283-286) with at expression in escherichia coli.

[0433.0.0.18] to [0435.0.0.18] sees that [0433.0.0.0] is to [0435.0.0.0]

[0436.0.0.18] sees [0436.0.0.0]

[0436.1.0.18] sees [0436.1.0.0]

[0437.0.5.18] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts (seeing [0437.0.5.5])

[0438.0.5.18] sees [0438.0.5.5]

[0438.1.5.18] sees [0438.0.5.5])

[0439.0.0.18] sees [0439.0.0.18]

[0439.1.5.18] sees [0439.0.5.5]

[0440.0.0.18] sees [0440.0.0.0]

[0440.1.5.18] sees [0440.0.5.5]

[0441.0.0.18] sees [0441.0.0.0]

[0442.0.0.18] sees [0442.0.0.0]

[0442.1.5.18] sees [0442.0.5.5]

[0443.0.0.18] to [0445.0.0.18] sees that [0443.0.0.0] is to [0445.0.0.0]

[0445.1.5.18] sees [0445.0.5.5]

[0445.1.9.18] to [0445.3.9.18] sees that [0445.1.9.9] is to [0445.3.9.9]

[0446.0.0.18] to [0451.0.0.18] sees that [0446.0.0.0] is to [0451.0.0.0]

[0451.1.5.18] sees [0451.0.5.5])

[00451.2.0.18] sees [00451.1.0.0]

[0452.0.0.18] to [0455.0.0.18] sees that [0452.0.0.0] is to [0455.0.0.0]

[0455.0.5.18] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example lipid acid) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being lipid or lipid acid) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

Except said process, as (1999) Proc.Natl.Acad.Sci.USA 96 (22) such as Cahoon: from vegetable material, extract plant lipid as described in (1986) Analytic Biochemistry 152:141-145 such as 12935-12940 and Browse.The qualitative and quantitative analysis of lipid or lipid acid is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:Oily Press (Oily Press Lipid Library; 2); Christie, William W., GasChromatography and Lipids.A Practical Guide-Ayr, Scotland:Oily Press, 1989, Repr.1992, IX, 307 pp. (Oily Press Lipid Library; 1); " Progress inLipid Research, Oxford:Pergamon Press, 1 (1952)-16 (1977), exercise question is Progress in the Chemistry of Fats and Other Lipids CODEN.(seeing [0455.0.5.5])

[0456.0.0.18] sees [0456.0.0.0]

[0457.0.18.18] embodiment 9: the purifying of Q9 or Coenzyme Q10 99.0

[0458.0.18.18] example is to analyze coenzyme.By using standard method of analysis, the HPLC (as The Journal of Lipid Research, the 39th rolls up, and 2099-2105 is described in 1998) that particularly has UV or Electrochemical Detection can clearly detect the existence of coenzyme product to the biology analysis of recombinating.

Produce and the possible method of preparation coenzyme such as Coenzyme Q10 99.0 for example has been described among WO2003056024, J57129695, J57202294, DE3416853 and the DD-229152.Other method that is used to separate each fine chemicals can also be at WO 9500634; Fat-Sci.Technol., (1992) 94,4,153-57; DD-294280; DD-293048; JP-145413; DD-273002; DD-271128; SU1406163; JP-166837; JP-176705; Acta-Biotechnol., (1986) 6,3,277-79; DD-229152; DE3416854; DE3416853; JP-202840; JP-048433; JP-125306; JP-087137; JP-014026; Find among WO2003056024 and the WO200240682.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

[0459.0.18.18] is common, uses acetone or hexane extraction coenzyme, and further purifying can be realized by the column chromatography of using suitable resin.

If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.18.18] depends on the fine chemicals that is produced in addition, can also use crystallization or distillation to come purifying.These two kinds of methods are that those skilled in the art are well-known.

[0461.0.18.18] embodiment 10: clone SEQ ID NO:22609 is used for expressing plant

[0462.0.0.18] sees [0462.0.0.0]

[0463.0.18.18] passes through pcr amplification SEQ ID NO:22609 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.18] to [0466.0.0.18] sees that [0464.0.0.0] is to [0466.0.0.0]

[0467.0.18.18] selects following primer sequence for genes of SEQ ID NO:22609:

I) forward primer (SEQ ID NO:22701)

atgggacaca?agcccttata?ccg

Ii) reverse primer (SEQ ID NO:22702)

ttatcgcgat?gattttcgct?gcg

[0468.0.0.18] to [0479.0.0.18] sees that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.18.18] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:22609

[0481.0.0.18] to [0513.0.0.18] sees that [0481.0.0.0] is to [0513.0.0.0]

The different plants that [0514.0.18.18] analyzed the results are shown in following table:

Table 1 metabolite profile information

??ORF	Metabolite	Method	??Min	??Max
					??YBR184W	Coenzyme Q10 99.0	??LC	??1.70	??1.95
??YDR513W	Q9	??LC	??1.31	??1.73
					??YER156C	Q9	??LC	??1.32	??1.58
??YER174C	Q9	??LC	??1.31	??1.72
					??YPR138C	Coenzyme Q10 99.0	??LC	??1.65	??3.57
??YPR172W	Q9	??LC	??1.34	??1.66
					??b3644	Q9	??LC	??1.37	??2.30
??b2426	Q9	??LC	??1.38	??1.77
					??b2703	Q9	??LC	??1.37	??2.18
??b2729	Q9	??LC	??1.35	??1.41
					??b0175	Q9	??LC	??1.45	??1.70
??b0730	Q9	??LC	??1.30	??3.03
					??b1829	Coenzyme Q10 99.0	??LC	??1.48	??5.33
??b2699	Coenzyme Q10 99.0	??LC	??1.62	??3.20
					??b2699	Q9	??LC	??1.34	??3.53
??b3605	Q9	??LC	??1.35	??1.41

[0515.0.0.18] to [0552.0.0.18] sees that [0515.0.0.0] to [0552.0.0.0] comprises [0530.1.0.0] to [0530.6.0.0] and [0552.2.0.0]

[0552.1.18.18] embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF_ title	Metabolite	??Min	??Max
				??b1829	Coenzyme Q10 99.0	??1.28	??1.65

Table 2 shows that Coenzyme Q10 99.0 has increased in the genetic modification maize plant of expressing e. coli k12 nucleotide sequence b1829.

In one embodiment, under the situation that Escherichia coli protein b1829 or its homologue are enhanced as the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue in maize plant, preferably, giving the fine chemicals Threonine is increased between 28% and 65%.

[0553.0.18.18]

1. produce the method for Coenzyme Q10 99.0 and/or Q9, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation Coenzyme Q10 99.0 and/or Q9.

2. produce the method for Coenzyme Q10 99.0 and/or Q9, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable, and described nucleic acid molecule is given in biology or its part Coenzyme Q10 99.0 and/or Q9 quantity increases;

B) contain just like Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of Coenzyme Q10 99.0 and/or Q9 quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of Coenzyme Q10 99.0 and/or Q9 quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that Coenzyme Q10 99.0 in biology or its part and/or Q9 quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives in biology or its part Coenzyme Q10 99.0 and/or Q9 quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part Coenzyme Q10 99.0 and/or Q9 quantity increases;

H) nucleic acid molecule, its coding contains just like Table IV, the polypeptide of consensus sequence shown in the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable and give biology or its part in Coenzyme Q10 99.0 and/or Q9 quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give Coenzyme Q10 99.0 and/or the increase of Q9 quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded Coenzyme Q10 99.0 and/or Q9.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or the bonded Coenzyme Q10 99.0 and/or the Q9 of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is as Table II the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, preferred Table II B the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 capable shown in polypeptide or its fragment, described nucleic acid molecule is given in biology or its part Coenzyme Q10 99.0 and/or Q9 quantity increases;

B) contain just like Table I the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 is capable, the nucleic acid molecule of preferred Table II B the 5th or 7 row, 231-234 and/or 235-242 and/or the 599-602 nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of Coenzyme Q10 99.0 and/or Q9 quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of Coenzyme Q10 99.0 and/or Q9 quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that Coenzyme Q10 99.0 in biology or its part and/or Q9 quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives in biology or its part Coenzyme Q10 99.0 and/or Q9 quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part Coenzyme Q10 99.0 and/or Q9 quantity increases;

H) nucleic acid molecule, its coding contains just like Table IV, the polypeptide of consensus sequence shown in the 7th row, 231-234 and/or 235-242 and/or 599-602 are capable and give biology or its part in Coenzyme Q10 99.0 and/or Q9 quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give Coenzyme Q10 99.0 and/or the increase of Q9 quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A the 5th or 7 row, 231-234 and/or 235-242 and/or 599-602 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, 231-234 and/or 235-242 and/or 599-602 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in Coenzyme Q10 99.0 and/or Q9 quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part Coenzyme Q10 99.0 and/or Q9 quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps Coenzyme Q10 99.0 and/or Q9 level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the Coenzyme Q10 99.0 that will measure and/or Q9 level or expression of polypeptides level and the standard Coenzyme Q10 99.0 of when described candidate compound or the sample that comprises described multiple compound lack, measuring and/or Q9 or expression of polypeptides level; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that Coenzyme Q10 99.0 in plant or the microorganism and/or Q9 produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of Coenzyme Q10 99.0 and/or Q9 quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of expression of polypeptides of Coenzyme Q10 99.0 in biology or its part and/or the increase of Q9 quantity and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that Coenzyme Q10 99.0 in the cell and/or Q9 produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that Coenzyme Q10 99.0 and/or Q9 increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce Coenzyme Q10 99.0 and/or Q9;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) Coenzyme Q10 99.0 and/or the Q9 level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give Coenzyme Q10 99.0 and/or the raising of Q9 level in the host cell after it is expressed with wild-type.

20. identify and to give the method that Coenzyme Q10 99.0 in the cell and/or Q9 produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that Coenzyme Q10 99.0 in biology or its part and/or Q9 quantity or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce Coenzyme Q10 99.0 and/or Q9;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) Coenzyme Q10 99.0 and/or the Q9 level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give Coenzyme Q10 99.0 and/or the raising of Q9 level in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that its nucleic acid molecule, right that contains claim 6 wants in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of water 14 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify the nucleic acid molecule of giving Coenzyme Q10 99.0 and/or Q9 increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control Coenzyme Q10 99.0 and/or Q9 level at biology.

25. makeup, medicine, nutritive compositions, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to protect Vegetable oil lipoprotein, oils, lipid or the wax that contains each fine chemicals.

27. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to produce oils, lipid, fat or the wax that contains each fine chemicals that is derived from microorganism.

28. the nucleic acid molecule of claim 6; the polypeptide of claim 14; the nucleic acid construct of claim 7; claim 8 or 9 carrier; antagonist or agonist according to claim 17 evaluation; the antibody of claim 15; the plant of claim 16 or plant tissue; the results material of claim 16; the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for the production for treating congestive heart failure; myocardial dysfunction; the thrombocyte size of dwindling; limited biologically active pdgf; the essential DCM (dilated cardiomyopathy); tired; pectoralgia; expiratory dyspnea and palpitaition; other performance of hypertension and cardiovascular disorder; the Coenzyme Q10 99.0 level that Q10 uses the HMG-CoA reductase inhibitor to cause reduces; ill gum; gingivitis; cancer; AIDS; other immune dysfunction; the muscular dystrophy relevant with heart trouble; tired out and the damage of the muscle relevant with exercise; fat; neurodegenerative disorders; male sterility; chronic stable angina pectoris; the plasma adiponectin levels of peroxide significantly reduces; skin photoage; fat peroxidation or dwindle the tumour size; alleviate metastatic breast cancer; improve sperm motility; be protected from oxidative stress; the pharmaceutical preparation of stabilizing cell membrane or protection seminal fluid; perhaps be used to produce the nutritional supplement of makeup or food or feed.

[0554.0.0.18] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.19] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.19] sees [0001.0.0.0] for disclosing of this paragraph.

[0002.0.19.19] plant produces several important secondary metabolites by the plain approach of phenylpropyl alcohol from phenylalanine.This type of material comprises flavonoid, xylogen, Weibull, Whitfield's ointment and hydroxyl cortex cinnamomi acid ester.Recently studies show that about the plain approach of phenylpropyl alcohol the biosynthetic traditional view of xylogen is incorrect.Although think that for a long time the hydroxylation of this approach and methylation reaction are to take place on free hydroxyl group styracin level, the enzyme of plain 3-hydroxylation of the phenylpropyl alcohol of proof catalysis now and 3-O-methylation reaction utilizes shikimic acid and CoA conjugates as substrate.Recently to the clone who participates in forulic acid and the biosynthetic acetaldehyde dehydrogenase of sinapinic acid show these two kinds of materials to small part by the formation (see Nair etc., 2004, Plant Cell, 16,544-554 and quoted passage thereof) of deriving of the oxygenizement of coniferyl aldehyde and sinapyladehyde.

[0003.0.0.19] forulic acid is to be found in the seed or leaf of most plants, particularly such as the material in the herbal wheat bran of wheat, paddy rice and avenaceous.The structure of its chemical structure and curcumine is closely similar, and curcumine is to give seasonings turmeric xanchromatic material.

The amount of forulic acid depends on species and growth conditions and changes in [0004.0.0.19] vegetable material; Therefore additive is that this kind material is originated more reliably than food or undressed draft material.

[0005.0.0.19] forulic acid has antioxidant properties, and this makes it become important anti-ageing additive, and these antioxidant properties also help other potential application of forulic acid.These are included in the application of diabetes, cardiovascular disorder, cancer, neuroprotective, bone sex change, climacterium, immunity and (perhaps) exercise performance aspect.

[0006.0.0.19] compares the interpolation forulic acid and can significantly reduce total cholesterol and triacylglycerol concentration in the blood in the male rat of hypercholesterolemia bait of throwing something and feeding with control group.Yet, add forulic acid and can increase HDL (' good cholesterol ').

As many other diet materials, forulic acid is that antioxidant-but it is a kind of extraordinary antioxidant.Forulic acid helps neutralization to be known as the free radical of ' super-oxide ', ' hydroxyl radical free radical ' and ' Nitrous Oxide ' especially.It can act synergistically with other antioxidant, gives their extra usefulness.In addition, can activate forulic acid even higher antioxidant activity by being exposed to the UV line, this shows that it helps to protect skin to avoid sun damage.

In microbiology was used, forulic acid was as the substrate of Vanillin (as for example being described in WO 9735999 or DE19960106) or melanochrome (WO 9720944) generation.

[0007.0.0.19] styracin comprises coffic acid and forulic acid, also is good antioxidant.Experiment has been found that these compounds can stop the growth of cancer cell.

In addition, sinapinic acid is the biosynthetic intermediate of syringyl lignin in angiosperm, and in some taxonomical groups also as the precursor of solubility secondary metabolites.Regulation and control (the Ruegger etc. that the biosynthesizing of mustard seed acid esters, sinapoylglucose, sinapoylmalate and sinapoylcholine and accumulation are grown in mouse ear mustard genus and other member of Rhaphanus at least, 1999,119 (1): 101-10,1999).

Because forulic acid or these favourable physiological roles of sinapinic acid and Agricultural biotechnologies potential, need to identify to participate in forulic acid or the metabolic enzyme of sinapinic acid or other proteinic gene, and generation has mutant or the transgenic plant strain of utilizing these genetic modification forulic acid or sinapinic acid content.

A kind of method that [0008.0.19.19] improves biosynthesizing throughput is to use recombinant DNA technology.Therefore, people's expectation can produce forulic acid or sinapinic acid in plant.The inhuman production of this type allows the most suitable and the most effective quality, quantity and the screening of producing biology of control.The latter is particularly important for commercial production economics, and also is favourable for the human consumer therefore.In addition, people are desirably in the plant and produce forulic acid or sinapinic acid, so that improve plant production power and to the resistance of biology as discussed previously and abiotic stress.

The recombinant DNA technology method has used some years with by increasing different biosynthesis genes and study the production that its influence to fine chemicals production improves fine chemicals in microorganism and the plant.For example, it is reported that the xenthophylls astaxanthin can result from the nectary of rotaring gene tobacco plant.Those transgenic plant are to prepare by the carrier transformation of tobacco plant that agrobacterium tumefaciens mediates, and wherein carrier comprises the ketolase encoding gene (called after crtO) from H.pluvialis, and the Pds gene of tomato is as the promotor and the leader sequence of encoding.Those results show that about 75% carotenoid that is found in the conversion plant flowers contains ketone group.

[0009.0.19.19] if can obtain producing algae, plant or other microorganism of a large amount of forulic acid or sinapinic acid, should be favourable therefore.The protokaryon or the eukaryotic microorganisms that relate to this type of conversion in some embodiments that the present invention is hereinafter discussed.

If it also should be favourable can obtaining root, leaf, stem, fruit or spend the middle plant that produces a large amount of forulic acid or sinapinic acid.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

The quality that [0010.0.19.19] therefore improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the forulic acid or the sinapinic acid that for example are present in as mentioned above in plant and some microorganisms are limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is specific forulic acid or sinapinic acid spectrum in the balanced diet as far as possible, because excessive forulic acid or sinapinic acid have negative effect on the specific concentrations.Further improving the quality only may be by adding other forulic acid or sinapinic acid, and these compositions all are limited.

[0011.0.19.19] is necessary with balance mode forulic acid or sinapinic acid to be added into suitable biology in order to guarantee the high quality of food and animal-feed.

[0012.0.19.19] therefore, still be starved of one or more codings and participate in forulic acid or the biosynthetic enzyme of sinapinic acid or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but need forever to improve the method that obtains the highest intrinsic energy forulic acid or sinapinic acid, on the other hand, reduce the byproduct that produces in the production process as far as possible.

[0013.0.0.19] sees [0013.0.0.0] for disclosing of this section.

[0014.0.19.19] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is forulic acid or sinapinic acid.Therefore, in the present invention, term " fine chemicals " is meant forulic acid or sinapinic acid as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of forulic acid or sinapinic acid as used herein.

[0015.0.19.19] an embodiment, term " fine chemicals " or " each fine chemicals " meaning are meant at least a have forulic acid or the active chemical compound of sinapinic acid.

In one embodiment, term " fine chemicals " meaning is meant forulic acid.In one embodiment, depend on the employed environment of term, term " fine chemicals " meaning is meant sinapinic acid.In whole specification sheets, term " fine chemicals " meaning is meant forulic acid or sinapinic acid, its salt, ester, thioesters or free form or be bonded to other compound such as the form of sugar or glycopolymers (as glucosides, for example bioside).

[0016.0.19.19] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0196, b0730, b1896, b2414, b3074, b3172, YBR184W, YDR513W or b2818 protein; With

(b) in allowing described biology, produce under the condition that fine chemicals is forulic acid or sinapinic acid and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has activity of proteins shown in Table II the 3rd row, 243-250 and 603 row, perhaps have by Table I the 5th or 7 row, 243-250 and 603 the row shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce under the condition that fine chemicals is forulic acid or sinapinic acid and make biological growth.

[0016.1.19.19] therefore, term " fine chemicals " meaning is meant and relevant " forulic acid " or its homologue of Table I the 243rd, 244,246,247, the 249 listed full sequences of row, and the meaning is meant and relevant " sinapinic acid " or its homologue of Table I the 245th, 248,250, the 6039 listed sequences of row.Therefore, term " fine chemicals " can refer to " forulic acid " or " sinapinic acid " according to environment and context.For the meaning of illustrating term " each fine chemicals " is meant " forulic acid " or " sinapinic acid " from the listed sequence of context, also can use term " each fine chemicals ".

[0017.0.0.19] and [0018.0.0.19] sees top paragraph [0017.0.0.0] and [0018.0.0.0] for disclosing of [0017.0.0.19] and [0018.0.0.19] these paragraphs

The method that [0019.0.19.19] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 243-250 and 603 row or by as Table I the 5th or 7 be listed as, the activity of proteins of the coded protein active of nucleic acid molecule is carried out above-mentioned modification shown in 243-250 and 603 row.

[0020.0.19.19] is surprisingly found out that, forulic acid or sinapinic acid (with regard to described protein and homologue and coding nucleic acid molecule (particularly shown in Table II the 3rd row, 243-250 and 603 row), being " fine chemicals " or " each fine chemicals ") content that e. coli k12 protein b0196, b0730, b1896, b2414, b3074, b3172, b2818 or yeast saccharomyces cerevisiae protein YBR184W or YDR513W protein are given the plant that transforms at transgene expression in the Arabidopis thaliana increase.

Above [0021.0.0.19] sees for disclosing of this paragraph [0021.0.0.0]

The sequence of [0022.0.19.19] e. coli k12 b0196 has been published in Blattner, F.R. etc., Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be to draw the synthetic instrumentality of acid.Therefore, in one embodiment, the inventive method comprises the purposes from e. coli k12 protein b0196 or its homologue as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases forulic acid, the preferred quantity of the forulic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b0196 is enhanced.

The sequence of e. coli k12 b0730 has been published in Blattner F.R. etc., Science277:1453-1474 (1997), and its activity to be defined as be that succinyl-coenzyme A synthase operon transcriptional and acyl are replied regulatory gene.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b0730 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases forulic acid, the preferred quantity of the forulic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b0730 is enhanced.

The sequence of e. coli k12 b1896 has been published in Blattner F.R. etc., Science277:1453-1474 (1997), and its activity to be defined as be that protein has the trehalose-6-phosphate synthase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b1896 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases sinapinic acid, the preferred quantity of the sinapinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b1896 is enhanced.

The sequence of b2414 e. coli k12 has been published in Blattner F.R. etc., Science277:1453-1474 (1997), and its function is defined as PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b2414 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases forulic acid, the preferred quantity of the forulic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b2414 is enhanced.

The sequence of e. coli k12 b3074 has been published in Blattner F.R. etc., Science277:1453-1474 (1997), and its activity be defined as be the supposition the tRNA synthetase protein.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b3074 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases forulic acid, the preferred quantity of the forulic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b3074 is enhanced.

The sequence of e. coli k12 b3172 has been published in Blattner F.R. etc., Science277:1453-1474 (1997), and its activity to be defined as be the protein with arginyl succsinic acid synthase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b3172 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases sinapinic acid, the preferred quantity of the sinapinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b3172 is enhanced.

The sequence of yeast saccharomyces cerevisiae YBR184W has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be unclassified protein.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YBR184W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases forulic acid, the preferred quantity of the forulic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of unclassified protein YBR184W is enhanced.

The sequence of yeast saccharomyces cerevisiae YDR513W has been published in Jacq, C. etc., Nature 387 (6632 supplementary issue), 75-78 (1997), and its activity to be defined as be to have the active protein of glutaredoxin (thiol transferase) (glutathione reductase).Therefore, in one embodiment, the inventive method comprises the purposes with described active protein YDR513W as shown here, it is used for producing each fine chemicals in biological or its part as described, particularly increases sinapinic acid, the preferred quantity of the sinapinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YDR513W is enhanced.

The sequence of e. coli k12 b2818 (accession number NP_417295) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be N-acetylglutamat synthase (amino acid N-Transacetylase).Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having the active gene product of amino-acid acetyltransferase acetylglutamate kinase superfamily, preferably having a purposes of the active protein of N-acetylglutamat synthase (amino acid N-Transacetylase) or its homologue, it is used for producing fine chemicals in biological or its part as described, be sinapinic acid, particularly increase sinapinic acid, the preferred quantity of the sinapinic acid of free or combining form.

The homologous compound (=homologue) of [0023.0.19.19] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, the 243rd, 244,246,247 or 249 row.Particularly, each fine chemicals in the biology is given in active raising, preferred ferulaic acid content increases.

In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, the 245th, 248,250 or 603 row.Particularly, each fine chemicals in the biology is given in active raising, preferred sinapinic acid content increases.

The homologue of [0023.0.19.19] polypeptide shown in Table II the 3rd row, 243-248,250 and 603 row can be by the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, 243-248,250 and 603 row or can be polypeptide shown in Table II the 7th row, 243-248,250 and 603 row.

The homologue of polypeptide shown in Table II the 3rd row, the 243rd, 244,246,247 row can be have increase ferulaic acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, the 243rd, 244,246,247 row or Table II the 7th row, the 243rd, 244,246,247 row.

The homologue of polypeptide shown in Table II the 3rd row, the 245th, 248,250,6039 row can be have increase sinapinic acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, the 245th, 248,250,6039 row or Table II the 7th row, the 245th, 248,250,6039 row.

The homologue of [0023.1.0.19] polypeptide shown in Table II the 3rd row, 243-250 and 603 row can be by the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, 243-250 and 603 row or can be polypeptide shown in Table II the 7th row, 243-250 and 603 row.

Above [0024.0.0.19] sees for disclosing of this paragraph [0024.0.0.0]

[0025.0.19.19] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause in biology or its part, the preferred described biomass cells forulic acid or sinapinic acid level to increase, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example activity of proteins shown in Table II the 3rd row, 243-250 and 603 row.In preferred embodiments, protein or polypeptide have proteinic above-mentioned extra activity shown in Table II the 3rd row, 243-250 and 603 row.In this manual, if this kind protein or polypeptide still have any one proteinic biology or the enzyme activity shown in Table II the 3rd row, 243-250 and 603 row, if promptly compare with any one e. coli k12 shown in Table II the 3rd row, 243-250 and 603 row or yeast saccharomyces cerevisiae protein, its have at least initial enzymic activity 10%, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as each fine chemicals of increase in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

Above [0025.1.0.19] and [0025.2.0.19] sees for disclosing of [0025.1.0.19] and [0025.2.0.19] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.19] to [0033.0.0.19] saw for disclosing of [0026.0.0.19] to [0033.0.0.19] these paragraphs

[0034.0.19.19] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, its have protein shown in Table II the 3rd row, 243-250 and 603 row or by the protein of nucleic acid molecule encoding shown in Table I the 5th row, 243-250 and 603 row or its homologue (homologue shown in Table I the 7th row, 243-250 and 603 row) active protein expression level or active aspect different, and its difference aspect biological chemistry or genetics reason.Therefore it shows each fine chemicals quantity that increases.

Paragraph [0035.0.0.0] was to [0044.0.0.0] above [0035.0.0.19] to [0044.0.0.19] saw for disclosing of [0035.0.0.19] to [0044.0.0.19] these paragraphs

[0045.0.19.19] in one embodiment, under the situation that the activity of e. coli k12 protein b0196 or its homologue (shown in Table II the 5th or 7 row, the 243rd row) is enhanced, give each fine chemicals, preferred forulic acid be increased in 10% and 25% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b0730 or its homologue (shown in Table II the 5th or 7 row, the 244th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred forulic acid be increased in 38% and 97% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b1896 or its homologue (shown in Table II the 5th or 7 row, the 245th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred sinapinic acid be increased in 38% and 98% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2414 or its homologue (shown in Table II the 5th or 7 row, the 246th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred forulic acid be increased in 34% and 86% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b3074 or its homologue (shown in Table II the 5th or 7 row, the 247th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred forulic acid be increased in 35% and 73% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b3172 or its homologue (shown in Table II the 5th or 7 row, the 248th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred sinapinic acid be increased in 31% and 89% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YBR184W or its homologue (shown in Table II the 5th or 7 row, the 249th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred forulic acid be increased in 30% and 37% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YDR513W or its homologue (shown in Table II the 5th or 7 row, the 250th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred sinapinic acid be increased in 30% and 39% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2818 or its homologue (shown in Table II the 5th or 7 row, the 603rd row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred sinapinic acid be increased in 27% and 54% or more between.

[0046.0.0.19] at e. coli k12 protein b0196 or its homologue, under the situation that the activity that for example can draw acid to synthesize instrumentality is enhanced, preferably gives the increase of fine chemicals forulic acid in one embodiment.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example succinic thiokinase operon transcriptional and acyl are replied under the situation that the activity of regulatory gene is enhanced, and preferably giving the fine chemicals forulic acid increases.

In one embodiment, at e. coli k12 protein b1896 or its homologue, for example under the situation that the activity of trehalose-6-phosphate synthase 5 is enhanced, preferably giving the fine chemicals sinapinic acid increases.

In one embodiment, at e. coli k12 protein b2414 or its homologue, for example under the situation that the activity of PLP dependent enzyme cysteine synthase A and O-acetylserine sulfhydrylase A subunit is enhanced, preferably giving the fine chemicals forulic acid increases.

In one embodiment, at e. coli k12 protein b3074 or its homologue, for example under the situation that the activity of Jia Ding tRNA synthetic enzyme is enhanced, preferably giving the fine chemicals forulic acid increases.

In one embodiment, at e. coli k12 protein b3172 or its homologue, for example have under the situation that the activity of proteins of arginyl succsinic acid synthase activity is enhanced, preferably giving the fine chemicals sinapinic acid increases.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YBR184W or its homologue is enhanced, preferably giving the fine chemicals forulic acid increases.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YDR513W or its homologue is enhanced, preferably giving the fine chemicals sinapinic acid increases.

In one embodiment, at e. coli k12 protein b2818 or its homologue, for example under the situation that the activity of N-acetylglutamat synthase (amino acid N-Transacetylase) is enhanced, preferably, give each fine chemicals, preferably give the fine chemicals sinapinic acid and increase.

[[0047.0.0.19] and [0048.0.0.19] sees top paragraph [0047.0.0.0] and [0048.0.0.0] for disclosing of [0047.0.0.19] and [0048.0.0.19] these paragraphs

[0049.0.19.19] has to give and improves each fine chemicals forulic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the 243rd, 244,246, the sequence of consensus sequence shown in 247 row or as Table II the 5th or 7 be listed as, the 243rd, 244,246, its function homologue described in polypeptide or the literary composition shown in 247 and/or 249 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 243rd, 244,246, nucleic acid molecule or its function homologue as herein described shown in 247 and/or 249 row) sequence of coded polypeptide, and have the activity that the forulic acid level of giving described in the literary composition increases.

Have to give and improve sinapinic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 7th row, the 245th, 248, the sequence of consensus sequence shown in 250 and/or 603 row and/or as Table II the 5th or 7 be listed as, the 245th, 248, its function homologue described in polypeptide or the literary composition shown in 250 and/or 603 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 245th, 248, nucleic acid molecule or its function homologue as herein described shown in 250 and/or 603 row) sequence of coded polypeptide, and have the activity that the sinapinic acid level of giving described in the literary composition increases.

[0050.0.19.19] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, the sylvite of forulic acid or sinapinic acid or sodium salt, perhaps their ester or glucoside (for example diglucoside).

[0051.0.19.19] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise the composition of forulic acid or sinapinic acid.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of forulic acid or sinapinic acid.

Above [0052.0.0.19] sees for disclosing of this paragraph [0052.0.0.0]

[0053.0.19.19] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, protein or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row) expression that described protein is given nucleic acid molecule encoding of the present invention increase, and have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row), perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by nucleic acid molecule of the present invention coded protein or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by nucleic acid molecule of the present invention coded protein or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row);

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) shown in Table II the 3rd row, 243-250 and 603 row).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.19.19] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improve coded protein or have shown in Table II the 3rd or 5 row, 243-250 and 603 row protein or its homologue (for example polypeptide shown in Table II the 5th or 7 row, 243-250 and 603 row) active expression of polypeptides or activity after give each fine chemicals and increase.

Paragraph [0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.19] to [0067.0.0.19] saw for disclosing of [0055.0.0.19] to [0067.0.0.19] these paragraphs

[0068.0.19.19] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of forulic acid or sinapinic acid.

Above [0069.0.0.19] sees for disclosing of this paragraph [0069.0.0.0]

[0070.0.19.19] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, the composition of for example favourable forulic acid and sinapinic acid or their biological chemistry derivative for example contains the composition of high level (from the physiology of nutrition angle) forulic acid and/or sinapinic acid or their derivative.

Above [0071.0.0.19] sees for disclosing of this paragraph [0071.0.0.0]

[0072.0.0.19]％

[0073.0.19.19] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of polypeptide of the present invention or its homologue (for example shown in Table II the 5th or 7 row, 243-250 and 603 row) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation.

[0074.0.19.19] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded.

Paragraph [0075.0.0.0] was to [0077.0.0.0] above [0075.0.0.19] to [0077.0.0.19] saw for disclosing of [0075.0.0.19] to [0077.0.0.19] these paragraphs

[0078.0.19.19] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes.

Method well known to those skilled in the art be can use and purified fermentation broth, tunning, plant or plant product come.For these different methods of setting up gradually, product is forulic acid or sinapinic acid or contains forulic acid and the composition of sinapinic acid, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

Paragraph [0075.0.0.0] was to [0084.0.0.0] above [0079.0.0.19] to [0084.0.0.19] saw for disclosing of [0079.0.0.19] to [0084.0.0.19] these paragraphs

[0085.0.19.19] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) nucleotide sequence or derivatives thereof shown in Table I the 5th or 7 row, 243-250 and 603 row, perhaps

(b) genetic regulatory element that effectively is connected with nucleotide sequence or derivatives thereof shown in Table I the 5th or 7 row, 243-250 and 603 row, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.19] and [0087.0.0.19] sees top paragraph [0086.0.0.0] and [0087.0.0.0] for disclosing of [0086.0.0.19] and [0087.0.0.19] these paragraphs

[0088.0.19.19] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Stronger and output is enhanced to the resistance of biological and abiotic stress because the nutritive value that for example is used for the plant of feeding animals such as poultry depends on above-mentioned fine chemicals or plant, so this is very important for the plant breeder.

Above [0088.1.0.19] sees for disclosing of this paragraph [0088.1.0.0]

Paragraph [0089.0.0.0] was to [0094.0.0.0] above [0089.0.0.19] to [0094.0.0.19] saw for disclosing of [0089.0.0.19] to [0094.0.0.19] these paragraphs

[0095.0.19.19] advantageously increases forulic acid or sinapinic acid pond to separate a large amount of pure basically fine chemicals and/or to obtain the resistance of the raising of biological and abiotic stress and obtain high yield by method of the present invention in genetically modified organism.

[0096.0.19.19] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.

Above [0097.0.0.19] sees for disclosing of this paragraph [0097.0.0.0]

[0098.0.19.19] in preferred embodiments, each fine chemicals be produce according to the present invention and carry out isolating where necessary.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.19.19].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition that comprises each fine chemicals.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.19.19] comprises the transgenic plant that the method according to this invention synthetic comprises fine chemicals such as forulic acid or sinapinic acid and can advantageously directly put on market, and do not need institute's synthetic fine chemicals is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet each fine chemicals that the method according to this invention produces can also be as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free fine chemicals from biology, advantageously separate from plant.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because forulic acid or sinapinic acid are positioned at cell in the microorganism, must relate to separation to their recovery to biomass.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.

[0101.0.19.19] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 11 27-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.19.19] for example, forulic acid or sinapinic acid can advantageously detect by HPLC, LC or GC separation method.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect the existence of the product that contains forulic acid or sinapinic acid to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

[0103.0.19.19] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide has the sequence shown in Table II the 5th or 7 row, 243-250 and 603 row; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) the preferred nucleic acid molecule that comprises the mature form nucleic acid molecule at least, described mature form nucleic acid molecule have the sequence shown in Table I the 5th or 7 row, 243-250 and 603 row;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer of sequence shown in Table III the 7th row, 243-250 and 603 row obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise consensus sequence with sequence shown in Table IV the 7th row, 243-250 and 603 row and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, 243-250 and 603 row; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.19.19] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 243-250 and 603 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor is not made up of sequence shown in Table I A the 5th or 7 row, 243-250 and 603 row.In one embodiment, the identity of sequence shown in the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 243-250 and 603 row is less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA the 5th or 7 row, 243-250 and 603 the row shown in polypeptide of sequence.

[00103.2.19.19] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 243-250 and 603 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor is not made up of sequence shown in Table I B the 5th or 7 row, 243-250 and 603 row.In one embodiment, the identity of sequence shown in the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 243-250 and 603 row is less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIB the 5th or 7 row, 243-250 and 603 the row shown in polypeptide of sequence.

[0104.0.19.19] in one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 243-250 and 603 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method is not made up of sequence shown in Table I the 5th or 7 row, 243-250 and 603 row.In one embodiment, the identity of sequence shown in nucleic acid molecule of the present invention and Table I the 5th or 7 row, 243-250 and 603 row is less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 243-250 and 603 the row shown in polypeptide of sequence.

Paragraph [0105.0.0.0] and [0107.0.0.0] above [0105.0.0.19] to [0107.0.0.19] sees for disclosing of [0105.0.0.19] to [0107.0.0.19] these paragraphs

Advantageously improved in [0108.0.19.19] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in 243-250 and 603 row, from Table II the 5th or 7 row, aminoacid sequence shown in 243-250 and 603 row is derived or is listed as from containing Table IV the 7th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 243-250 and 603 row, perhaps its coding has as Table II the 3rd, 5 or 7 row, the enzymic activity of polypeptide or bioactive polypeptide shown in 243-250 and 603 row or for example to give each fine chemicals behind its expression or active the increasing be derivative or the homologue that forulic acid or sinapinic acid increase.

[0109.0.19.19] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make each fine chemicals, particularly forulic acid or sinapinic acid that the inventive method produced be optimized synthetic.

[0110.0.0.19] helps the nucleic acid molecule of the polypeptide that the inventive method and coding have the used polypeptide active of the used or of the present invention process of the inventive method (for example shown in Table II the 5th or 7 row, 243-250 and 603 row protein or by the protein of nucleic acid molecule encoding or its homologue shown in Table I the 5th or 7 row, 243-250 and 603 row (shown in Table II the 5th or 7 row, 243-250 and 603 row)) and can determine from generally open database.

Above [0111.0.0.19] sees for disclosing of this paragraph [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.19.19] the inventive method is the isolated nucleic acid sequences form, its coding has the polypeptide of polypeptide active shown in Table II the 3rd row, 243-250 and 603 row or has the polypeptide of peptide sequence shown in Table II the 5th and 7 row, 243-250 and 603 row, and gives forulic acid or the increase of sinapinic acid level.

Paragraph [0113.0.0.0] and [0120.0.0.0] above [0113.0.0.19] to [0120.0.0.19] sees for disclosing of [0113.0.0.19] to [0120.0.0.19] these paragraphs

[0121.0.19.19] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, peptide sequence or its functional homologue as herein described have the difference of one or more amino acid moleculars shown in 243-250 and 603 row, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, the 243rd, 244,246,247, giving the forulic acid level after the activity of peptide sequence shown in 249 row increases, and is perhaps improving Table II the 5th or 7 row, the 245th, 248, giving the sinapinic acid level after the activity of peptide sequence shown in 250 and 603 row increases.

Paragraph [0122.0.0.0] and [0127.0.0.0] above [0122.0.0.19] to [0127.0.0.19] sees for disclosing of [0122.0.0.19] to [0127.0.0.19] these paragraphs

The synthetic oligonucleotide primer thing that [0128.0.19.19] polymerase chain reaction (PCR) amplification is used (for example the primer shown in Table III the 7th row, 243-250 and 603 row to) can be based on sequence shown in this paper, for example sequence shown in Table I the 5th or 7 row, 243-250 and 603 row or produce from sequence deutero-sequence shown in Table II the 5th or 7 row, 243-250 and 603 row.

[0129.0.19.19] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence is from described comparison shown in Table IV the 7th row, 243-248,250 and 603 row.

[0130.0.19.19] sees [0130.0.0.0] for disclosing of this paragraph.

Paragraph [0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.19] to [0138.0.0.19] saw for disclosing of [0131.0.0.19] to [0138.0.0.19] these paragraphs

[0139.0.19.19] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with for the Table I the 5th of forulic acid or 7 row, the 243rd, 244,246,247,249 row, preferred Table I B the 5th or 7 row, the 243rd, 244,246,247, sequence shown in 249 row or for the Table I the 5th of sinapinic acid or 7 row, the 245th, 248,250,6039 row, preferred Table I B the 5th or 7 row, the 245th, 248,250, sequence hybridization shown in 6039 row, and the coding expression has each fine chemicals, the i.e. active peptide of forulic acid or sinapinic acid of increasing.

Paragraph [0140.0.0.0] and [0146.0.0.0] above [0140.0.0.19] to [0146.0.0.19] sees for disclosing of [0140.0.0.19] to [0146.0.0.19] these paragraphs

[0147.0.19.19] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.Be meant these molecules with one of nucleotide sequence shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 5th or 7 row, 243-250 and 603 row complementary nucleic acid molecule: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.19.19] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 5th or 7 row, nucleotide sequence or its portion homologous are at least about 30% shown in 243-250 and 603 row, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases forulic acid or sinapinic acid.

[0149.0.19.19] nucleic acid molecule of the present invention comprises nucleotide sequence, one of nucleotide sequence shown in described sequence and Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 5th or 7 row, 243-250 and 603 row or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (increasing as giving forulic acid or sinapinic acid) and the protein of protein active shown in Table II the 5th row, 243-250 and 603 row, preferred Table II B the 5th or 7 row, 243-250 and 603 row randomly.

[00149.1.19.19] randomly, in one embodiment, have another or various active with the nucleotide sequence of one of nucleotide sequence shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 5th or 7 row, 243-250 and 603 row hybridization, described activity is known activity or is used for these protein of note for the protein shown in Table II the 3rd row, 243-250 and 603 row, preferred Table II B the 5th or 7 row, 243-250 and 603 row.

[0150.0.19.19] in addition, nucleic acid molecule of the present invention can only contain the part of the coding region of one of nucleotide sequence shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 5th or 7 row, 243-250 and 603 row, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give forulic acid or sinapinic acid increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with the antisense sequences of one of sequence shown in sense strand, Table I the 5th or 7 row, 243-250 and 603 row of one of sequence shown in Table I the 5th or 7 row, 243-250 and 603 row or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.

Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.To produce fragment or its gene product of polynucleotide sequence shown in Table I the 5th or 7 row, 243-250 and 603 row with the right PCR of primer shown in Table III the 7th row, 243-250 and 603 row.Preferably Table I B the 7th is listed as, 190-226 is capable or 564-594 is capable.

[0151.0.0.19]: see top [0151.0.0.0] for disclosing of this paragraph

[0152.0.19.19] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and the abundant homology of aminoacid sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising forulic acid as be shown in the examples (the 243rd, 244,246,247,249 row) or sinapinic acid (the 245th, 248,250,6039 row) level.

[0153.0.19.19] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, the amino-acid residue that described aminoacid sequence comprises with the aminoacid sequence minimal number is identical or of equal value shown in Table II the 5th or 7 row, 243-250 and 603 row (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, protein or its part have the activity of polypeptide shown in Table II the 3rd row for example as herein described, 243-250 and 603 row shown in Table II the 5th or 7 row, 243-250 and 603 row.

[0154.0.19.19] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein is at least about 30%, 35%, 45% or 50% with complete amino acid sequence homology shown in Table II the 5th or 7 row, 243-250 and 603 row, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.19] and [0156.0.0.19] sees top paragraph [0155.0.0.0] and [0156.0.0.0] for disclosing of [0155.0.0.19] and [0156.0.0.19] these paragraphs

[0157.0.19.19] the present invention relate in addition owing to the genetic code degeneracy be different from one of nucleotide sequence (with its part) shown in Table I the 5th or 7 row, 243-250 and 603 row and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase), for example comprise shown in Table IV the 7th row, 243-248,250 and 603 row polypeptide of sequence or as Table II the 5th or 7 row, 243-250 and 603 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment shown in Table IV the 7th row, 243-248,250 and 603 row consensus sequence or shown in Table II the 5th or 7 row, 243-250 and 603 row the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise shown in Table IV the 7th row, 243-248,250 and 603 row consensus sequence or as Table II the 5th or 7 row, 243-250 and 603 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise the sequence shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I A the 5th or 7 row, 243-250 and/or 603 row.Preferably, nucleic acid molecule of the present invention is a functional homologous compound or identical with nucleic acid molecule shown in Table I B the 5th or 7 row, 243-250 and/or 603 row.

Paragraph [0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.19] to [0160.0.0.19] saw for disclosing of [0158.0.0.19] to [0160.0.0.19] these paragraphs

[0161.0.19.19] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (nucleic acid molecule that for example comprises sequence shown in Table I the 5th or 7 row, 243-250 and 603 row) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

Above [0162.0.0.19] sees for disclosing of this paragraph [0162.0.0.0]

[0163.0.19.19] preferably, under stringent condition with the nucleic acid molecule of the present invention of sequence hybridization shown in Table I the 5th or 7 row, 243-250 and 603 row corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals quantity increase in biology or its part (for example tissue, cell or cell chamber) after using activity of proteins).

Above [0164.0.0.19] sees for disclosing of this paragraph [0164.0.0.0]

[0165.0.19.19] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence (for example sequence shown in Table I the 5th or 7 row, 243-250 and 603 row) of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method.

[0166.0.0.19] and [0167.0.0.19] sees top paragraph [0166.0.0.0] and [0167.0.0.0] for disclosing of [0166.0.0.19] and [0167.0.0.19] these paragraphs

[0168.0.19.19] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are different with the contained sequence of sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with aminoacid sequence shown in Table II the 5th or 7 row, 243-250 and 603 row at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 row, 243-250 and 603 the row shown in sequence identical at least about 60%, more preferably identical at least about 70% with one of sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, even more preferably with as Table II the 5th or 7 row, 243-250 and 603 the row shown in sequence at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 row, 243-250 and 603 the row shown in sequence identical at least about 96%, 97%, 98% or 99%.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.The contained sequence of these amino acid sequence of polypeptide and sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row is different, but has kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with aminoacid sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 is identical at least about 60% with the sequence shown in 603 row, more preferably with as Table II the 5th or 7 be listed as, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 is identical at least about 70% with one of sequence shown in 603 row, even more preferably with as Table II the 5th or 7 be listed as, 243-250 and 603 row, preferred Table II B the 7th row, sequence shown in 243-250 and 603 row is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 243-250 and 603 row, preferred Table II B the 7th row, sequence shown in 243-250 and 603 row is at least about 96%, 97%, 98% or 99% is identical.

Paragraph [0169.0.0.0] was to [0172.0.0.0] above [0169.0.0.19] to [0172.0.0.19] saw for disclosing of [0169.0.0.19] to [0172.0.0.19] these paragraphs

[0173.0.19.19] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:24071 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:24071 sequence before use.

[0174.0.0.19]: see top [0174.0.0.0] for disclosing of this paragraph

[0175.0.19.19] for example, the sequence that has 80% homology at protein level and SEQ ID NO:24072 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:24072 sequence.

[0176.0.19.19] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtains shown in 243-250 and 603 row be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in 243-250 and 603 row, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in 243-250 and 603 row has essentially identical character and discerns.

[0177.0.19.19] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the functional equivalent that obtains of nucleotide sequence shown in 243-250 and 603 row be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in 243-250 and 603 row, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the polypeptide of the essentially identical character of polypeptide shown in 243-250 and 603 row.

Above [0178.0.0.19] sees for disclosing of this paragraph [0178.0.0.0]

[0179.0.19.19] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly shown in Table I the 5th or 7 row, 243-250 and 603 row), and thereby replace, add or disappearance and produce the nucleic acid molecule of coding as the homologue of protein sequence shown in Table II the 5th or 7 row, 243-250 and 603 go to introducing one or more amino acid in the coded protein.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to shown in Table I the 5th or 7 row, 243-250 and 603 row, introducing sudden change in the encoding sequence.

Paragraph [0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.19] to [0183.0.0.19] saw for disclosing of [0180.0.0.19] to [0183.0.0.19] these paragraphs

[0184.0.19.19] is employed to have as Table I the 5th or 7 row, the nucleotide sequence homologous compound of sequence shown in 243-250 and 603 row, perhaps come Table II the 5th or 7 row freely, 243-250 and 603 row, preferred Table II B the 7th row, the homologous compound of nucleotide sequence of sequence shown in 243-250 and 603 row also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from sequence shown in Table I the 5th or 7 row, 243-250 and 603 row or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.19.19] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more sequences shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 7th row, 243-250 and 603 row.In one embodiment, the preferred nucleic acid molecule comprises other Nucleotide the least possible, that do not show in the arbitrary sequence shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 7th row, 243-250 and 603 row.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method is identical with sequence shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 7th row, 243-250 and 603 row.

The employed one or more nucleic acid molecule encodings of [0186.0.19.19] also preferred the inventive method comprise polypeptide of sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method is identical with sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row.

[0187.0.19.19] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row polypeptide of sequence and contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, employed nucleic acid molecule is identical with the encoding sequence of coding sequence shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row in the method.

[0188.0.19.19] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active comparing with polypeptide expressed activity shown in Table II the 5th or 7 row, 243-250 and 603 row and under the same conditions do not reduced substantially.In one embodiment, polypeptide of the present invention is to comprise shown in Table II B the 7th row, 243-250 and 603 row sequence or by its homologue of forming.

[0189.0.19.19] be the homologous compound of sequence shown in Table I the 5th or 7 row, 243-250 and 603 row, or the homologue of the sequence of deutero-shown in Table II the 5th or 7 row, 243-250 and 603 row also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.19], [0191.0.0.19], [00191.1.0.19] and [0192.0.0.19] to [0203.0.0.19] is for [0190.0.0.19], [0191.0.0.19], [0191.1.0.19] and [0192.0.0.19] be paragraph [0190.0.0.0] above disclosing of [0203.0.0.19] these paragraphs seen, [0191.0.0.0], [0191.1.0.0] and [0192.0.0.0] is to [0203.0.0.0]

[0204.0.19.19] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding polypeptide or its segmental nucleic acid molecule shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row of mature form at least, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly forulic acid (the 243rd, 244,246,247,249 row) or sinapinic acid (the 245th, 248,250,6039 row) quantity increase;

(b) comprise, preferably comprise nucleic acid molecule or its segmental nucleic acid molecule shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 7th row, 243-250 and 603 row of mature form at least, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by use primer or the nucleic acid molecule of primer to increasing and obtain shown in Table III the 7th row, 243-250 and 603 row from cDNA library or genomic library, and in biological or its part, give each fine chemicals, i.e. the increase of forulic acid (the 243rd, 244,246,247,249 row) or sinapinic acid (the 245th, 248,250,603 row) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like consensus sequence shown in Table IV the 7th row, 243-248,250 and 603 row and give each fine chemicals in biological or its part, i.e. the nucleic acid molecule of forulic acid (the 243rd, 244,246,247,249 row) or sinapinic acid (the 245th, 248,250,6039 row) quantity increase;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be the nucleic acid molecule that forulic acid (the 243rd, 244,246,247,249 row) or sinapinic acid (the 245th, 248,250,6039 row) quantity increase, the structural domain of the polypeptide of described peptide coding shown in Table II the 5th or 7 row, 243-250 and 603 row, preferred Table II B the 7th row, 243-250 and 603 row; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, nucleic acid molecule shown in 243-250 and 603 row or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 243-250 and 603 row, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from sequence shown in Table I A or IB the 5th or 7 row, 243-250 and 603 row by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention is not made up of the sequence shown in Table I A or IB the 5th or 7 row, 243-250 and 603 row.In another embodiment, nucleic acid molecule of the present invention is identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99% with sequence at least 30% shown in Table I A or IB the 5th or 7 row, 243-250 and 603 row.In another embodiment, the nucleic acid molecule peptide sequence shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row of not encoding.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 243-250 and 603 row at least one or the different polypeptide of a plurality of amino acid, and the protein of shown in Table II A or IIB the 5th or 7 row, 243-250 and the 603 row sequence of therefore not encoding.Therefore, in one embodiment, therefore, in one embodiment, (a) do not form to the coded protein of the nucleotide sequence of (l) by sequence shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 243-250 and 603 row at least 30% identical and with sequence shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.19] and [0206.0.0.19] sees top paragraph [0205.0.0.0] and [0206.0.0.0] for disclosing of [0205.0.0.19] and [0206.0.0.19] these paragraphs

[0207.0.19.19] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of glutamic acid metabolism, phosphoenolpyruvic acid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

Paragraph [0208.0.0.0] was to [0226.0.0.0] above [0208.0.0.19] to [0226.0.0.19] saw for disclosing of [0208.0.0.19] to [0226.0.0.19] these paragraphs

[0227.0.19.19] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 243-250 and the 603 capable sequence or derivatives thereofs of mentioning, can advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one L-glutamic acid or phosphoenolpyruvic acid pathways metabolism in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with one or more sequences shown in Table I the 5th or 7 row, 243-250 and 603 row with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.19.19]％

[0229.0.19.19] can with the sequence of employed other the favourable nucleotide sequences that the combination of sequence and/or aforementioned biosynthesis gene is expressed shown in Table I the 5th or 7 row, 243-250 and 603 row of present method for other gene of coding aromatic amino acid (as tryptophane, phenylalanine or tyrosine) approach.These genes cause the synthetic increase of essential amino acids tryptophan, phenylalanine or tyrosine.

Above [0230.0.0.19] sees for disclosing of this paragraph [0230.0.0.0]

[0231.0.19.19] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded forulic acid or sinapinic acid simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.For example it be known to those skilled in the art that, to the inhibition of the enzyme of degraded forulic acid and/or sinapinic acid or check and to cause in the plant forulic acid or sinapinic acid accumulation to increase.

Paragraph [0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.19] to [0276.0.0.19] saw for disclosing of [0232.0.0.19] to [0276.0.0.19] these paragraphs

[0277.0.19.19] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.

Paragraph [0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.19] to [0282.0.0.19] saw for disclosing of [0278.0.0.19] to [0282.0.0.19] these paragraphs

[0283.0.19.19] in addition, can from cell, separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example resist protein shown in Table II the 3rd row, 243-250 and 603 row, the perhaps antibody of polypeptide or its antigen part shown in Table II the 5th or 7 row, 243-250 and 603 row, it can utilize polypeptide of the present invention or its fragment to produce by standard technique.Monoclonal antibody preferably.

Above [0284.0.0.19] sees for disclosing of this paragraph [0284.0.0.0]

[0285.0.19.19] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 243-250 and 603 the row shown in sequence or by as Table I the 5th or 7 row, 243-250 and 603 the row shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.19.19] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, 243-248, consensus sequence shown in 250 and 603 row or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, 243-248, consensus sequence or by its polypeptide of forming shown in 250 and 603 row, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can be by arbitrary aminoacid replacement.

Paragraph [0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.19] to [0290.0.0.19] saw for disclosing of [0287.0.0.19] to [0290.0.0.19] these paragraphs

[0291.0.19.19] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from sequence shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in 243-250 and 603 row, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in 243-250 and 603 row.In another embodiment, described polypeptide of the present invention be can't help shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row sequence and is formed.

Above [0292.0.0.19] sees for disclosing of this paragraph [0292.0.0.0]

[0293.0.19.19] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention has by one or more amino acid and other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row.In another embodiment, polypeptide of the present invention be can't help shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row sequence and is formed.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide is not made up of the coded sequence of nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 243-250 and 603 row.

[0294.0.19.19] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, the polypeptide of activity of proteins shown in 243-250 and 603 row, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in 243-250 and 603 row.

Paragraph [0295.0.0.0] was to [0297.0.0.0] above [0295.0.0.19] to [0297.0.0.19] saw for disclosing of [0295.0.0.19] to [0297.0.0.19] these paragraphs

The chemical of [00297.1.0.19] non-polypeptide of the present invention is for example not have the activity of polypeptide shown in Table II the 3rd, 5 or 7 row, 243-250 and 603 row and/or the polypeptide of aminoacid sequence.

[0298.0.19.19] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 243-250 and 603 the row shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used has and the identical aminoacid sequence of sequence shown in Table II the 5th or 7 row, 243-250 and 603 row.

[0299.0.19.19] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% with one of nucleotide sequence sequence homology shown in Table I the 5th or 7 row, 243-250 and 603 row, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises nucleotide sequence or the coded aminoacid sequence of its homologue with nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, 243-250 and 603 row.

[0300.0.19.19] therefore, and be described in detail as this paper, and polypeptide of the present invention is because natural variation or mutagenesis can be different with sequence shown in Table II the 5th or 7 row, 243-250 and 603 row on aminoacid sequence.Therefore, the complete amino acid sequence homology that this polypeptide contains with sequence shown in Table II A or IIB the 5th or 7 row, 243-250 and 603 row is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

Above [0301.0.0.19] sees for disclosing of this paragraph [0301.0.0.0]

The biologically-active moiety of [0302.0.19.19] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence shown in 243-250 and 603 row or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

Above [0303.0.0.19] sees for disclosing of this paragraph [0303.0.0.0]

[0304.0.19.19] operation nucleic acid molecule of the present invention may cause generation have basically the active of shown in Table II the 3rd row, 243-250 and 603 row polypeptide and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

Paragraph [0305.0.0.0-[0308.0.0.0] above [0305.0.0.19] to [0308.0.0.19] sees for disclosing of [0305.0.0.19] to [0308.0.0.19] these paragraphs

[0309.0.19.19] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, protein shown in 243-250 and 603 row is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " other polypeptide " in 243-250 and 603 row is meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, polypeptide shown in 243-250 and 603 row is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, proteinic and shown in 243-250 and 603 row from the protein of identical or different biology.In one embodiment, " other polypeptide " that is not shown in Table II the 5th or 7 row, 243-250 and 603 row do not given each fine chemicals increase in biology or its part.

Paragraph [0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.19] to [0334.0.0.19] saw for disclosing of [0310.0.0.19] to [0334.0.0.19] these paragraphs

[0335.0.19.19] confirmed that the dsRNAi method to the expression that reduces nucleotide sequence shown in Table I the 5th or 7 row, 243-250 and 603 row and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change by the expression that reduces shown in Table I the 5th or 7 row, 243-250 and 603 row nucleotide sequence and/or its homologue.Double stranded rna molecule at the coded protein expression of the nucleotide sequence that is used for reducing shown in Table I the 5th or 7 row, 243-250 and 603 row sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

Paragraph [0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.19] to [0342.0.0.19] saw for disclosing of [0336.0.0.19] to [0342.0.0.19] these paragraphs

[0343.0.19.19] as describing, in order to cause effective reduction of expression, at dsRNA and shown in Table I the 5th or 7 row, 243-250 and 603 row between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that sequence or its homologue begin to produce shown in Table I the 5th or 7 row, 243-250 and 603 row in a kind of organism.

Paragraph [0344.0.0.0] was to [0361.0.0.0] above [0344.0.0.19] to [0361.0.0.19] saw for disclosing of [0344.0.0.19] to [0361.0.0.19] these paragraphs

[0362.0.19.19] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, polypeptide shown in 243-250 and 603 row, for example coding has protein such as Table II the 3rd row, the polypeptide of polypeptide active shown in 243-250 and 603 row) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Transgenosis with polypeptide of polypeptide active shown in Table II the 5th or 7 row, 243-250 and 603 row is meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, the activity that is noted as polypeptide shown in Table II the 3rd row, 243-250 and 603 row (for example having polypeptide of sequence shown in Table II the 5th or 7 row, 243-250 and 603 row) is enhanced.Example and the inventive method are described in above.

Above [0363.0.0.19] sees for disclosing of this paragraph [0363.0.0.0]

[0364.0.19.19] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---promotor of for example naturally occurring coding gene of polypeptide of the present invention shown in Table II the 3rd row, 243-250 and 603 row and combination of protein coding sequence shown in Table I the 5th row, 243-250 and 603 row accordingly---becomes transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.19] to [0373.0.0.19] for [0365.0.0.19], disclosing of these paragraphs of-[0373.0.0.19] sees that top paragraph [0365.0.0.0] is to [0373.0.0.0]

[0374.0.19.19] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, forulic acid that produces in the inventive method or sinapinic acid, particularly each fine chemicals also can be from plant with free forulic acid or each fine chemicals of sinapinic acid, particularly free or be bonded to compound or the isolated in form of part (for example glucoside, for example diglucoside).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.19] and [0376.0.0.19] sees top paragraph [0375.0.0.0] and [0376.0.0.0] for disclosing of [0375.0.0.19] and [0376.0.0.19] these paragraphs

[0377.0.19.19] therefore the invention still further relates to the method according to this invention, separated the forulic acid or the sinapinic acid that are produced thus.

[0378.0.19.19] can separate by this way and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than 90% the forulic acid or the sinapinic acid that produce in the method.As required, resultant forulic acid or sinapinic acid can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.19.19] in one embodiment, forulic acid or sinapinic acid are the mixtures of each fine chemicals.

The suitable synthetic parent material of forulic acid that [0380.0.19.19] obtains in the method for the invention or sinapinic acid as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises forulic acid that separation produces or sinapinic acid composition or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention be the forulic acid that produces of the inventive method or sinapinic acid or genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine purposes or be used to produce the purposes of forulic acid or sinapinic acid, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

[0381.0.0.19] to [0384.0.0.19] for [0381.0.0.19], disclosing of these paragraphs of-[0384.0.0.19] sees that top paragraph [0381.0.0.0] is to [0384.0.0.0]

The fermented liquid that [0385.0.19.19] obtains with this kind approach particularly contains the fermented liquid with other organic acid, amino acid, polypeptide or polysaccharide blended forulic acid or sinapinic acid, and dry matter content accounts for the 1-70% of weight, preferred 7.5 to 25% usually.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means and to utilize the concentration of sugar during this period of time to remain on or be reduced to 0-10g/l in the fermention medium, preferred to 0-3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.19.19] therefore can also be further purified the forulic acid or the sinapinic acid that produce according to the present invention.For this reason, the composition that will contain product separates (by for example open column chromatography or HPLC), and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

Paragraph [0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.19] to [0392.0.0.19] saw for disclosing of [0387.0.0.19] to [0392.0.0.19] these paragraphs

[0393.0.19.19] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify and the nucleic acid molecule of the present invention nucleic acid molecule of under lax stringent condition, hybridizing of the nucleic acid molecule shown in Table I the 5th or 7 row, 243-250 and 603 row, preferred Table I B the 5th or 7 row, 243-250 and 603 row particularly, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

Paragraph [0394.0.0.0] was to [0398.0.0.0] above [0394.0.0.19] to [0398.0.0.19] saw for disclosing of [0394.0.0.19] to [0398.0.0.19] these paragraphs

[0399.0.19.19] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving each fine chemicals generation raising under these conditions.

[00399.1.19.19] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect and whether depends on the active of polypeptide shown in Table II the 5th or 7 row, 243-250 and 603 row or its homologue or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high shown in Table II the 5th or 7 row, 243-250 and 603 row the biology phenotype more much at one of activity of proteins.

Paragraph [0400.0.0.0] was to [0416.0.0.0] above [0400.0.0.19] to [0416.0.0.19] saw for disclosing of [0400.0.0.19] to [0416.0.0.19] these paragraphs

[0417.0.19.19] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor to forulic acid or sinapinic acid biosynthetic pathway has resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block forulic acid or sinapinic acid synthetic inhibitor.

Paragraph [0418.0.0.0] was to [0423.0.0.0] above [0418.0.0.19] to [0423.0.0.19] saw for disclosing of [0418.0.0.19] to [0423.0.0.19] these paragraphs

[0424.0.19.19] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other organic acids.

Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

Paragraph [0425.0.0.0] was to [0434.0.0.0] above [0425.0.0.19] to [0434.0.0.19] saw for disclosing of [0425.0.0.19] to [0434.0.0.19] these paragraphs

[0435.0.19.19] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.19.19] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce forulic acid or sinapinic acid.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.19.19] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.19.19] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.19.19] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.19.19] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad 50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

Paragraph [0441.0.0.0] was to [0443.0.0.0] above [0441.0.0.19] to [0443.0.0.19] saw for disclosing of [0441.0.0.19] to [0443.0.0.19] these paragraphs

[0444.0.19.19] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.19.19] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

Paragraph [0446.0.0.0] was to [0450.0.0.0] above [0446.0.0.19] to [0450.0.0.19] saw for disclosing of [0446.0.0.19] to [0450.0.0.19] these paragraphs

[0451.0.5.19], [0452.0.0.19] and [0453.0.0.19] are for [0451.0.5.19], and top [0451.0.5.5], [0452.0.0.0] and [0453.0.0.0] are seen in disclosing of [0452.0.0.19] and [0453.0.0.19] these paragraphs

[0454.0.19.19] analyzing nucleic acid molecules is to the influence of forulic acid or sinapinic acid generation

[0455.0.19.19] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example forulic acid or sinapinic acid) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being forulic acid or sinapinic acid) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of IndustrialChemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and MolecularBiology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery andpurification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", JohnWiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recoveryprocesses for biological Materials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques inbiotechnology ", Noyes Publications).

Above [0456.0.0.19] sees for disclosing of this paragraph [0456.0.0.0]

[0457.0.19.19] embodiment 9: forulic acid or sinapinic acid purifying

[0458.0.19.19] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS or TLC can clearly detect the existence of forulic acid or sinapinic acid to the biology analysis of recombinating.

It is biological to use the following step to analyze, as the total amount that produces in the inventive method used yeast: by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 4022S1V diluter with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.10ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, used Hewlett Packard 1100HPLC, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Technologies, can be from Ultra Scientific Inc., 250 Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6 * 250mm[Waters, 34 Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

[0459.0.19.19] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified forulic acid or sinapinic acid.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

Above [0460.0.0.19] sees for disclosing of this paragraph [0460.0.0.0]

[0461.0.19.19] embodiment 10: clone SEQ ID NO:24071,24083,24177,24353,24865,25117,25357,25361 or 92604 is used for expressing plant

Above [0462.0.0.19] sees for disclosing of this paragraph [0462.0.0.0]

[0463.0.19.19] passes through pcr amplification SEQ ID NO:24071,24083,24177,24353,24865,25117,25357,25361 or 92604 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

Paragraph [0464.0.0.0] was to [0466.0.0.0] above [0464.0.0.19] to [0466.0.0.19] saw for disclosing of [0464.0.0.19] to [0466.0.0.19] these paragraphs

[0466.1.0.19] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.19.19] selects following primer sequence for genes of SEQ ID NO:24071:

I) forward primer (SEQ ID NO:24081)

atgcgtgctt?taccgatctg?ttta

Ii) reverse primer (SEQ ID NO:24082)

ttatttcgcc?gtaatgttaa?gcgcag

Select following primer sequence for genes of SEQ ID NO:24083:

I) forward primer (SEQ ID NO:24175)

atgggacaca?agcccttata?ccg

Ii) reverse primer (SEQ ID NO:24176)

ttatcgcgat?gattttcgct?gcg

Select following primer sequence for genes of SEQ ID NO:24177:

I) forward primer (SEQ ID NO:24351)

atgagtcgtt?tagtcgtagt?atcta

Ii) reverse primer (SEQ ID NO:24152)

ttacgcaagc?tttggaaagg?tagc

Select following primer sequence for genes of SEQ ID NO:24353:

I) forward primer (SEQ ID NO:24863)

atgagtaaga?tttttgaaga?taac

Ii) reverse primer (SEQ ID NO:24864)

ttactgttgc?aattctttct?cagtg

Select following primer sequence for genes of SEQ ID NO:24865:

I) forward primer (SEQ ID NO:25115)

atggaaaccg?tggcttacgc?tg

Ii) reverse primer (SEQ ID NO:24116)

ttatacgacg?cgtacgcccg?c

Select following primer sequence for genes of SEQ ID NO:25117:

I) forward primer (SEQ ID NO:25355)

atgacgacga?ttctcaagca?tctc

Ii) reverse primer (SEQ ID NO:25356)

ttactggcct?ttgttttcca?gattc

Select following primer sequence for genes of SEQ ID NO:25357:

I) forward primer (SEQ ID NO:25359)

atgtaccaaa?ataatgtatt?gaatgct

Ii) reverse primer (SEQ ID NO:25360)

tcaatagtgc?attaactctc?ccatt

Select following primer sequence for genes of SEQ ID NO:25361:

I) forward primer (SEQ ID NO:25495)

atggagacca?atttttcctt?cgact

Ii) reverse primer (SEQ ID NO:25496)

ctattgaaat?accggcttca?atattt

Select following primer sequence for genes of SEQ ID NO:92604:

I) forward primer (SEQ ID NO:92658)

atggtaaagg?aacgtaaaac?cgagt

Ii) reverse primer (SEQ ID NO:92659)

ttaccctaaa?tccgccatca?acac

Paragraph [0468.0.0.0] was to [0470.0.0.0] above [0468.0.0.19] to [0470.0.0.19] saw for disclosing of [0468.0.0.19] to [0470.0.0.19] these paragraphs

The PCR product that [0470.1.19.19] utilizes the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBIFermentas) phosphorylation to produce by Pfu Turbo archaeal dna polymerase, and the clone enters in the binary vector of treated mistake.

Above [0471.0.0.19] sees for disclosing of this paragraph [0471.0.0.0]

[0471.1.19.19] uses Pfu Turbo archaeal dna polymerase benefit flat in the second step building-up reactions by the DNA end of the PCR product that the Herculase archaeal dna polymerase produces.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Utilize the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBI Fermentas) phosphorylation PCR product, and be cloned in the carrier of handling.

Paragraph [0472.0.0.0] was to [0479.0.0.0] above [0472.0.0.19] to [0479.0.0.19] saw for disclosing of [0472.0.0.19] to [0479.0.0.19] these paragraphs

[0480.0.19.19] embodiment 11: express the generation of SEQ ID NO:24071,24083,24177,24353,24865,25117,25357,25361 or 92604 transgenic plant

Paragraph [0481.0.0.0] was to [0513.0.0.0] above [0481.0.0.19] to [0513.0.0.19] saw for disclosing of [0481.0.0.19] to [0513.0.0.19] these paragraphs

[0514.0.19.19] alternatively, as Mattila, P. and Kumpulainen J., J.Agric FoodChem.2002 Jun 19; 50 (13): detect forulic acid described in the 3660-7.

Alternatively, as Noda, M. and Matsumoto, M., Biochim Biophys Acta.1971Feb 2; 231 (1): detect sinapinic acid described in the 131-3.

The different plants of being analyzed the results are shown in following table 1:

Table 1

?ORF	Metabolite	Method	?Min	?Max
					?b0196	Forulic acid	LC	?1.10	?1.25
?b0730	Forulic acid	LC	?1.38	?1.97
					?b1896	Sinapinic acid	GC	?1.38	?1.98
?b2414	Forulic acid	LC	?1.34	?1.86
					?b3074	Forulic acid	LC	?1.35	?1.73
?b3172	Sinapinic acid	GC	?1.31	?1.89
					?YBR184W	Forulic acid	LC	?1.30	?1.37
?YDR513W	Sinapinic acid	GC	?1.30	?1.39

?b2818

Sinapinic acid

??GC

??1.27

??1.54

The 2nd row have shown metabolite forulic acid or the sinapinic acid of being analyzed.The 4th row and the 5th row have shown the ratio of metabolite between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

Paragraph [0515.0.0.0] was to [0530.0.0.0] above [0515.0.0.19] to [0530.0.0.19] saw for disclosing of [0515.0.0.19] to [0530.0.0.19] these paragraphs

Paragraph [0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.19] to [0530.6.0.19] saw for disclosing of [0530.1.0.19] to [0530.6.0.19] these paragraphs

Paragraph [0531.0.0.0] was to [0552.0.0.0] above [0531.0.0.19] to [0552.0.0.19] saw for disclosing of [0531.0.0.19] to [0552.0.0.19] these paragraphs

[0552.1.0.19]％

Above [0552.2.0.19] sees for disclosing of this paragraph [0552.2.0.0]

[0553.0.19.19]

1. produce the method for forulic acid or sinapinic acid, it comprises:

(a) in the activity that improves or produce shown in Table II the 5th or 7 row, 243-250 and 603 row protein or its function equivalent in non-human being or its one or more parts; With

(b) in allowing described biology, cultivate this biology under the condition of generation forulic acid or sinapinic acid.

2. produce the method for forulic acid or sinapinic acid, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, 243-250 and 603 row, and described nucleic acid molecule is given forulic acid or the increase of sinapinic acid quantity in biology or its part;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row, 243-250 and 603 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of forulic acid or sinapinic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of forulic acid or sinapinic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that forulic acid in biology or its part or sinapinic acid quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 243-250 and 603 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives in biology or its part forulic acid or sinapinic acid quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part forulic acid or sinapinic acid quantity increases;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 243-250 and 603 row and give biology or its part in forulic acid or sinapinic acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give forulic acid or the increase of sinapinic acid quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded forulic acid or sinapinic acid.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or the bonded forulic acid or the sinapinic acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, 243-250 and 603 row, and described nucleic acid molecule is given forulic acid or the increase of sinapinic acid quantity in biology or its part;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row, 243-250 and 603 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of forulic acid or sinapinic acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of forulic acid or sinapinic acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that forulic acid in biology or its part or sinapinic acid quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 243-250 and 603 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives in biology or its part forulic acid or sinapinic acid quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part forulic acid or sinapinic acid quantity increases;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 243-250 and 603 row and give biology or its part in quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give forulic acid or the increase of sinapinic acid quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from sequence shown in Table I A the 5th or 7 row, 243-250 and 603 row by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or Table II A the 5th or 7 be listed as, 1-5 is capable and/or 334-338 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in forulic acid or sinapinic acid quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part forulic acid or sinapinic acid quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps forulic acid or sinapinic acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) give the flat or expression of polypeptides level of sour water and the standard forulic acid of when described candidate compound or the sample that comprises described multiple compound lack, measuring or sinapinic acid or expression of polypeptides level by the forulic acid that will measure or mustard and relatively identify agonist or antagonist; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that forulic acid in plant or the microorganism or sinapinic acid produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of forulic acid or sinapinic acid quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of expression of polypeptides of forulic acid in biology or its part or the increase of sinapinic acid quantity and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that forulic acid in the cell or sinapinic acid produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that forulic acid or sinapinic acid increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce forulic acid or sinapinic acid;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) forulic acid or the sinapinic acid level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give forulic acid or the raising of sinapinic acid level in the host cell after it is expressed with wild-type.

20. identify and to give the method that forulic acid in the cell or sinapinic acid produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that forulic acid in biology or its part or sinapinic acid quantity or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce forulic acid or sinapinic acid;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) forulic acid or the sinapinic acid level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare that it gives the host after expressing with wild-type

Forulic acid or sinapinic acid level improve in the cell.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify the nucleic acid molecule of giving forulic acid or sinapinic acid increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control forulic acid or sinapinic acid level at biology.

25. agrochemicals, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of any one described method of claim 1-5, claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein fine chemicals is forulic acid or sinapinic acid.

[0554.0.0.19] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.20] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.20] sees [0001.0.0.0]

The lipid acid that [0002.0.20.20] plant produces unusual long-chain is docosoic acid (C22:0), Lignoceric acid (C24:0), cerinic acid (C26:0) and/or myricyl acid (C30:0) for example.

Very-long-chain fatty acid (VLCFA) is to extend complex body by the film conjugated fatty acid (to extend enzyme, FAE) utilize acetyl-CoA substrate synthetic.First reaction of extending relates to malonyl CoA and the condensation of long-chain substrate produces beta-keto acyl-CoA.Subsequent reaction is the reductive action of β-hydroxyl acyl-CoA, the dehydration of alkene acyl CoA, is that reduction reaction forms the acetyl-CoA that extends for the second time afterwards.Has keying action aspect the chain length of the fatty acids products that the beta-keto acyl-CoA synthase (KCS) of catalyzing and condensing reaction is found in determining seed oils, and be the plain enzyme (Lassner etc. of limit that seed VLCFA produces, Plant Cell, 8 (1996), 281-292).

The extension process can be carried out repetition to produce the member of 20,22 and 24 carbon chain lengths.Although this type of very-long-chain fatty acid is the less important member of body adipose membrane, they exercise valuable function undoubtedly, obviously help stabilizing membrane, particularly those films in the peripheral nerve cell.

Docosoic acid (22:0) mountain Yu acid is the composition of rapeseed oil (as many as 2%) and peanut oil (1-5%).

Docosoic acid is used to give the hair thermoregulation agent and wetting agent is kept its slick characteristic.

Lignoceric acid (24:0) (Lignoceric acid) is the composition of rapeseed oil (as many as 1%) and peanut oil (1-3%).

Cerinic acid (26:0) (hexacosanoic acid) is the mellisic composition.

Echinacea purpurea (Echinacea angustifolia) extract is sold as the natural health product that comprises the very-long-chain fatty acid cerinic acid.

Cerinic acid is used for makeup as the hair style product.

Myricyl acid (C30:0) (triacontanoic acid) is the mellisic composition.

Beeswax (cera alba) is to obtain from the secreted product of some body of gland that honeybee forms honeycomb.It is by fusion and the centrifugal solid impurity (cera flava) of having removed.At last, its full bleaching is become white (cera alba).Beeswax comprises the paraffin oil carbohydrate of 10-15%, the C16-C36 fatty acid ester of 35-37% and about 15% cerinic acid, myricyl acid and homologous compound thereof.Beeswax can be used as thickening material and the wetting Agent for Printing Inks in ointment, emulsifiable paste, lipstick and the manufacturing of other makeup, and can be used for skin-protection product as emulsifying agent, tenderizer, wetting agent and membrane-forming agent.

Beeswax also is used for the generation of candle.

Wax is general term; be used in reference to the mixture of the nonpolar lipid of long-chain; the nonpolar lipid of these long-chains forms the protective layer (keratin in the stratum corneum) on plant leaf and the fruit, and is present in animal (the stratum corneum lipid of the wax of honeybee, insect, the spermaceti of Physter macrocephalus, skin lipid, the uropygial gland of birds, the stored fat of flotation crustaceans), algae, fungi and the bacterium.

Many waxes that occurring in nature is found have commercial use in lubricating oil, foods and cosmetics industry.Proposed the resource of Jojoba oil for a long time, because this desert shrub is unusual at its generation wax rather than triacylglycerol (TAG) aspect the ability of seed storage lipid as the wax of supposition.These waxes be very-long-chain fatty acid and Fatty Alcohol(C12-C14 and C12-C18) ester (Miwa, 1971, J Am OilChem Soc 48,259-264).Owing to be mainly used in the productive expense height of the Simmondsia chinensis wax of cosmetic applications, still need the through engineering approaches crop plants so that in its seed oil, produce high-caliber wax ester.

The plant air surface is covered by cuticular wax, and cuticular wax is unusual long-chain (C ₂₀-C ₃₄) complex mixture of lipid acid, hydro carbons, aldehydes, ketone and ester.Except getting rid of atmosphere moisture content, they also prevent drying, and therefore be important arid resistance decision thing (Riederer and Schreiber, 2001, J.Exp.Bot 52,2023-2032).Except the abiotic stress resistance, the wax layer still is the part that the plant antibiont stress (particularly as for example Marcell and Beattie, 2002, Mol PlantMicrobe Interact.15 (12), the described insect of 1236-44) defence.In addition, they also give pollen particles stability, thus influence fertility and breeding.

Very-long-chain fatty acid (VLCFA) comprises more than 18 carbon atoms acid as docosoic acid, Lignoceric acid, cerinic acid and myricyl acid, is to keep the blodynamic neccessary composition of higher plant.The biosynthetic key enzyme of VLCFA, enzyme is extended in the acid of plastid external fat, is the main target site of chloracetamide herbicide.Along with fatty acid component and metabolic analysis, determined the reductive action of VLCFA in vivo to 14C labelled precursor (stearic acid, propanedioic acid, acetic acid).The restraining effect proof VLCFA biosynthetic first of recombinant protein and rate-limiting step-acetyl-CoA and malonyl CoA-beta-keto acyl-CoA condensation-be the main target spot (I50=10-100nM) of chlor(o)acetamide.The concentration of VLCFA is low in the untreated cell, and very long-chain compound mainly is found in plasma membrane lipid and the cuticular wax.Lipid acid be converted into than short chain or even very the long-chain composition to eliminate fully be to suppress the biosynthetic result of VLCFA.Particularly, the shortage of plasma membrane VLCFA relates to the poisonous plant effect of chlor(o)acetamide, and for example film takes place and mitotic restraining effect (Matthes, B., http://www.ub.uni-konstanz.de/kops/volltexte/2001/661/).

The wax production that improves in transgenic plant is by Broun etc., and 2004, Proc Natl.Acad.Sci, 101,4706-4711 reports.The author crosses expression transcriptional activation agent WIN1 in mouse ear mustard belongs to, the wax that is loaded with on the organ (arial organs) that causes ventilating increases.Since this cause variation complicated in the wax spectrum and transgenosis cross the expression plant carry g and D aspect characteristic change (Broun etc., 2004, Proc Natl.Acad.Sci, 101,4706-4711), still need the generation of regulatable more definite VLCFA.

The very long chain aliphatic alcohol that obtains from vegetable wax and beeswax has also been reported and can reduce plasma cholesterol in human body, and available data is supported VLCFA at peroxysome cholesterol metabolic to be had regulating effect and change LDL absorption and metabolism and (is discussed in Hargrove etc., 2004, Exp Biol Med (Maywood), 229 (3): 215-26).

Because docosoic acid (C22:0), Lignoceric acid (C24:0), cerinic acid (C26:0) and these favourable physiological roles of myricyl acid (C30:0) and nutrition, makeup and Agricultural biotechnologies potential, need to identify to participate in docosoic acid, Lignoceric acid, cerinic acid or the metabolic enzyme of myricyl acid or other proteinic gene, and generation has mutant or the transgenic plant strain of utilizing these genetic modification docosoic acids, Lignoceric acid, cerinic acid or myricyl acid content.

[0003.0.0.20]％

[0004.0.0.20]％

[0005.0.0.20]％

[0006.0.0.20]％

[0007.0.0.20]％

A kind of method that [0008.0.20.20] improves biosynthesizing throughput is to use recombinant DNA technology.Therefore, people's expectation can produce docosoic acid, Lignoceric acid, cerinic acid or myricyl acid in plant.This type production allows the most suitable and the most effective quality, quantity and the screening of producing biology of control.The latter is particularly important for commercial production economics, and also is favourable for the human consumer therefore.In addition, people are desirably in and produce docosoic acid, Lignoceric acid, cerinic acid or myricyl acid in the plant, so that improve plant production power and to the resistance of biology as discussed previously and abiotic stress.

The recombinant DNA technology method has used some years with by increasing different biosynthesis genes and study the production that its influence to fine chemicals production improves fine chemicals in microorganism and the plant.For example, it is reported that the xenthophylls astaxanthin can result from the nectary of rotaring gene tobacco plant.Those transgenic plant are to prepare by the carrier transformation of tobacco plant that agrobacterium tumefaciens mediates, and wherein carrier comprises the ketolase encoding gene (called after crtO) from H.pluvialis, and the Pds gene of tomato is as the promotor and the leader sequence of encoding.Those results show that about 75% carotenoid that is found in the conversion plant flowers contains ketone group.

[0009.0.20.20] if can obtain producing algae, plant or other microorganism of a large amount of docosoic acids, Lignoceric acid, cerinic acid or myricyl acid, should be favourable therefore.The protokaryon or the eukaryotic microorganisms that relate to this type of conversion in some embodiments that the present invention is hereinafter discussed.

If it also should be favourable can obtaining root, leaf, stem, fruit or spend the middle plant that produces a large amount of docosoic acids, Lignoceric acid, cerinic acid or myricyl acid.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

The quality that [0010.0.20.20] therefore improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid that for example are present in as mentioned above in plant and some microorganisms are limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is specific docosoic acid, Lignoceric acid, cerinic acid or myricyl acid spectrum in the balanced diet as far as possible, because excessive docosoic acid, Lignoceric acid, cerinic acid or myricyl acid have negative effect on the specific concentrations.Further improving the quality only may be by adding other docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, and these compositions all are limited.

[0011.0.20.20] is necessary with balance mode docosoic acid, Lignoceric acid, cerinic acid or myricyl acid to be added into suitable biology in order to guarantee the high quality of food and animal-feed.

[0012.0.20.20] therefore, still be starved of one or more codings and participate in docosoic acid, Lignoceric acid, cerinic acid or the biosynthetic enzyme of myricyl acid or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, two specific being even more important.On the one hand, but need forever to improve the method that obtains the highest intrinsic energy docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, on the other hand, reduce the byproduct that produces in the production process as far as possible.

[0013.0.0.20] sees [0013.0.0.0]

[0014.0.20.20] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.Therefore, in the present invention, term " fine chemicals " is meant docosoic acid, Lignoceric acid, cerinic acid or myricyl acid as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid as used herein.

In one embodiment, term " fine chemicals " meaning is meant docosoic acid.In one embodiment, depend on the context that uses it, term " fine chemicals " meaning is meant Lignoceric acid.In another embodiment, term " fine chemicals " meaning is meant cerinic acid.In another embodiment, term " fine chemicals " meaning is meant myricyl acid.In whole specification sheets, term " fine chemicals " meaning is meant docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, its salt, ester, thioesters or free form or is bonded to other compound such as the form of sugar or glycopolymers (as glucosides, for example bioside).

[0016.0.20.20] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0019, b0880, b1886, b1896, b3938, YDR513W, YER156C, YGL205W, YHR201C, YLR255C, YPR138C, b0255 protein; With

(b) in allowing described biology, produce under the condition that fine chemicals is docosoic acid, Lignoceric acid, cerinic acid or myricyl acid and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has activity of proteins shown in Table II A or IIB the 3rd row, 251-261,627 row, perhaps have by nucleic acid molecule encoded polypeptide sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row and

(b) in allowing described biology, produce under the condition that fine chemicals is docosoic acid, Lignoceric acid, cerinic acid or myricyl acid and make biological growth.

[0016.1.20.20] therefore, term " fine chemicals " meaning is meant and Table I A or relevant " docosoic acid " or its homologue of IB the 258th listed full sequence of row, and the meaning is meant and Table I A or IB the 251st, 255,259,261 or relevant " Lignoceric acid " or its homologue of the 627 listed sequences of row, and the meaning is meant and Table I A or IB the 256th, 257 or relevant " myricyl acid " of the 260 listed sequences of row.Therefore, term " fine chemicals " can refer to " docosoic acid ", " Lignoceric acid ", " cerinic acid " or " myricyl acid " according to environment and context.For being meant, the meaning of illustrating term " each fine chemicals " " docosoic acid ", " Lignoceric acid ", " cerinic acid " or " myricyl acid " from the listed sequence of context also can use term " each fine chemicals ".

[0017.0.0.20] to [0018.0.0.20]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.20.20] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II A or IIB the 3rd row, 251-261, protein active shown in 627 row or by as Table I A or IB the 5th or 7 be listed as, 251-261, the activity of proteins of the coded protein active of nucleic acid molecule is carried out above-mentioned modification shown in 627 row.

[0020.0.20.20] is surprisingly found out that, e. coli k12 protein b0019, b0880, b1886, b1896, b3938, b0255 or yeast saccharomyces cerevisiae protein YDR513W, YER156C, YGL205W, YHR201C, YLR255C, YPR138C transgene expression in Arabidopis thaliana is given the docosoic acid of the plant that transforms, Lignoceric acid, cerinic acid or myricyl acid are (with regard to described protein and homologue thereof and coding nucleic acid molecule (particularly as Table II A or IIB the 3rd row, 251-261, shown in 627 row), be " fine chemicals " or " each fine chemicals ") the content increase.

[0021.0.0.20] sees [0021.0.0.0]

The sequence of [0022.0.20.20] e. coli k12 b0019 has been published in Blattner, F.R. etc., Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be the protein with Na+/H+ antiport protein-active.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b0019 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases Lignoceric acid and/or cerinic acid, preferably free or the Lignoceric acid of combining form and/or the quantity of cerinic acid.In one embodiment, in the methods of the invention, the activity of protein b0019 is enhanced.

The sequence of e. coli k12 b0880 has been published in Blattner, F.R. etc., Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be the protein with dna replication dna inhibitor activity of stress-induced.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b0880 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases cerinic acid, the preferred quantity of the cerinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b0880 is enhanced.

The sequence of e. coli k12 b1886 has been published in Blattner, F.R. etc., Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be methyl acceptor chemotactic protein matter II, aspartic acid transmitter acceptor.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b1886 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases cerinic acid, the preferred quantity of the cerinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b1886 is enhanced.

The sequence of e. coli k12 b1896 has been published in Blattner, F.R. etc., Science277 (5331), 1453-1474 (1997), and its activity to be defined as be to have the active protein of trehalose-6-phosphate synthase.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b1896 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases cerinic acid, the preferred quantity of the cerinic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b1896 is enhanced.

The sequence of e. coli k12 b3938 has been published in Blattner, F.R. etc., and Science277 (5331), 1453-1474 (1997), and its activity is defined as the biosynthetic transcription repressor of methionine(Met).Therefore, in one embodiment, the inventive method comprises as shown here from the protein b3938 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases Lignoceric acid, the preferred quantity of the Lignoceric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b3938 is enhanced.

The sequence of yeast saccharomyces cerevisiae YDR513W has been published in Jacq, C. etc., Nature 387 (6632 supplementary issue), 75-78 (1997), and its activity to be defined as be the protein with glutathione reductase activity.Therefore, in one embodiment, the inventive method comprises the protein YDR513W as shown here or the purposes of its homologue, and it is used for producing each fine chemicals in biological or its part as described, particularly increases myricyl acid, the preferred quantity of the myricyl acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YDR513W is enhanced.

The sequence of yeast saccharomyces cerevisiae YER156C has been published in Dietrich, F.S. etc., and Nature 387 (6632 supplementary issue), 78-81 (1997), and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises the protein YER156C as shown here or the purposes of its homologue, and it is used for producing each fine chemicals in biological or its part as described, particularly increases myricyl acid, the preferred quantity of the myricyl acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YER156C is enhanced.

The sequence of yeast saccharomyces cerevisiae YGL205W has been published in Tettelin, H. etc., Nature 387 (6632 supplementary issue), 81-84 (1997), and its activity to be defined as be to have the active protein of ACOD.Therefore, in one embodiment, the inventive method comprises the protein YGL205W as shown here or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases docosoic acid, the preferred quantity of the docosoic acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YGL205W is enhanced.

The sequence of yeast saccharomyces cerevisiae YHR201C has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be the protein with Tripyrophosphoric acid 5 prime excision enzyme activity.Therefore, in one embodiment, the inventive method comprises the protein YHR201C as shown here or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases Lignoceric acid and/or cerinic acid, preferably free or the Lignoceric acid of combining form and/or the quantity of cerinic acid.In one embodiment, in the methods of the invention, the activity of protein YHR201C is enhanced.

The sequence of yeast saccharomyces cerevisiae YLR255C has been published in the EMBL Data Library, and February 1995, and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises the protein YLR255C as shown here or the purposes of its homologue, and it is used for producing each fine chemicals in biological or its part as described, particularly increases myricyl acid, the preferred quantity of the myricyl acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YLR255C is enhanced.

The sequence of yeast saccharomyces cerevisiae YPR138C has been published in Bussey, H. etc., Nature 387 (6632 supplementary issue), 103-105 (1997), and its activity to be defined as be to have the active protein of NH4+ transporter.Therefore, in one embodiment, the inventive method comprises the protein YPR138C as shown here or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases Lignoceric acid, the preferred quantity of the Lignoceric acid of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YPR138C is enhanced.

The sequence of e. coli k12 b0255 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be " CP4-6 prophage; IS911 homologue ".Therefore, in one embodiment, the inventive method comprises described intestinal bacteria " CP4-6 prophage; The IS911 homologue " purposes of proteinic or its homologue; it is used for producing fine chemicals in biological or its part as described; be linolic acid or Lignoceric acid and/or comprise linolic acid or the triglyceride level of Lignoceric acid, lipid, oils and/or fat, particularly increases linolic acid or Lignoceric acid and/or comprises linolic acid and/or the triglyceride level of Lignoceric acid, lipid, oils and/or fat, preferably free or the linolic acid of combining form or the quantity of Lignoceric acid.In one embodiment, described in the methods of the invention " CP4-6 prophage; The IS911 homologue " activity of proteins is enhanced or produces, for example from colibacillary " CP4-6 prophage; The IS911 homologue " activity of protein or its homologue is enhanced or produces.

The homologous compound (=homologue) of [0023.0.20.20] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, any one homologue of polypeptide is to have same or similar active homologue shown in Table II A or IIB the 3rd row, the 251st, 255,259,261 and 627 row.Particularly, each fine chemicals in the biology is given in active raising, preferred tetracosane acid content increases.

In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II A or IIB the 3rd row, the 251st, 252,253,254 and 259 row.Particularly, each fine chemicals in the biology is given in active raising, the preferred wax acid content increases.

In one embodiment, any one homologue of polypeptide is to have same or similar active homologue shown in Table II A or IIB the 3rd row, the 256th, 257 and 260 row.Particularly, each fine chemicals in the biology is given in active raising, preferred myricyl acid content increases.

In one embodiment, any one homologue of polypeptide is to have same or similar active homologue shown in Table II A or IIB the 3rd row, the 258th row.Particularly, each fine chemicals in the biology is given in active raising, preferred docosane acid content increases.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 251st, 255,259,261 and 627 row can be have increase the tetracosane acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 251st, 255,259,261 and 627 row or Table II A or IIB the 7th row, the 251st, 255,259,261 and 627 row.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 251st, 252,253,254 and 259 row can be have increase cerinic acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 251st, 252,253,254 and 259 row or Table II A or IIB the 7th row, the 251st, 252,253,254 and 259 row.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 256th, 257 and 260 row can be have increase myricyl acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 256th, 257 and 260 row or Table II A or IIB the 7th row, the 256th, 257 and 260 row.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 258th row can be have increase the docosane acid content and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 258th row or Table II A or IIB the 7th row, the 258th row.

[0024.0.0.20] sees [0024.0.0.0]

[0025.0.20.20] is according to the present invention, docosoic acid, Lignoceric acid, cerinic acid or myricyl acid level increase in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have activity of proteins shown in Table II A or IIB the 3rd row, 251-261,627 row.In preferred embodiments, protein or polypeptide have proteinic above-mentioned extra activity shown in Table II A or IIB the 3rd row, 251-261,627 row.In this manual, if this kind protein or polypeptide still have Table II A or IIB the 3rd row, 251-261, any one proteinic biology or the enzyme activity shown in 627 row, if promptly with Table II A or IIB the 3rd row, shown in e. coli k12 protein or 256-261 shown in 251-255 is capable are capable in the yeast saccharomyces cerevisiae protein any one compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I A or IB the 4th row and is expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as increase each fine chemicals in biological or its part.For example play eozoan and express biologically, and play eozoan with biological shown in Table I A or IB the 4th row from identical section, order, guiding principle or door from different sections, order, guiding principle or door.

[0025.1.0.20] sees [0025.1.0.0]

[0026.0.0.20] to [0033.0.0.20]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.20.20] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, its have protein shown in Table II A or IIB the 3rd row, 251-261,627 row or by the protein of nucleic acid molecule encoding shown in Table I A or IB the 5th row, 251-261,627 row or its homologue (as Table I A or IB the 7th row, 251-261,627 capable shown in homologue) active protein expression level or active aspect difference, and its difference aspect biological chemistry or genetics reason.Therefore it shows each fine chemicals quantity that increases.

[0035.0.0.20] to [0038.0.0.20]: see that [0035.0.0.0] is to [0038.0.0.0]

[0039.0.0.20]: see [0039.0.0.0]

[0040.0.0.20] to [0044.0.0.20]: see that [0040.0.0.0] is to [0044.0.0.0]

[0045.0.20.20] in one embodiment, under the situation that the activity of e. coli k12 protein b0019 or its homologue (shown in Table II A or IIB the 5th or 7 row, the 251st row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred cerinic acid be increased in 40% and 150% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b0019 or its homologue (shown in Table II A or IIB the 5th or 7 row, the 251st row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Lignoceric acid be increased in 46% and 224% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b0880 or its homologue (shown in Table II A or IIB the 5th or 7 row, the 252nd row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred cerinic acid be increased in 172% and 294% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b1886 or its homologue (shown in Table II A or IIB the 5th or 7 row, the 253rd row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred cerinic acid be increased in 39% and 335% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b1896 or its homologue (shown in Table II A or IIB the 5th or 7 row, the 254th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred cerinic acid be increased in 39% and 75% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b3938 or its homologue (shown in Table II A or IIB the 5th or 7 row, the 255th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Lignoceric acid be increased in 37% and 89% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YDR513W or its homologue (shown in Table II A or IIB the 5th or 7 row, the 256th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred myricyl acid be increased in 31% and 108% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YER156C or its homologue (shown in Table II A or IIB the 5th or 7 row, the 257th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred myricyl acid be increased in 33% and 73% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YGL205W or its homologue (shown in Table II A or IIB the 5th or 7 row, the 258th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred docosoic acid be increased in 74% and 132% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YHR201C or its homologue (shown in Table II A or IIB the 5th or 7 row, the 259th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred cerinic acid be increased in 46% and 227% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YHR201C or its homologue (shown in Table II A or IIB the 5th or 7 row, the 259th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Lignoceric acid be increased in 29% and 271% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR255C or its homologue (shown in Table II A or IIB the 5th or 7 row, the 260th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred myricyl acid be increased in 34% and 88% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YPR138C or its homologue (shown in Table II A or IIB the 5th or 7 row, the 261st row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Lignoceric acid be increased in 92% and 160% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b0255 or its homologue (shown in Table II A or IIB the 5th or 7 row, the 627th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Lignoceric acid be increased in 35% and 115% or more between.

[0046.0.0.20]％

[0047.0.0.20] to [0048.0.0.20]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.20.20] has to give and improves each fine chemicals Lignoceric acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:27297 as SEQ ID, 27298 or 27309,27310 or 27322,27323,27324 or 27325,27326,27327,27328,97769, shown in 97770 or Table IV the 7th row, the 251st, 255,259, the sequence of consensus sequence shown in 261 and 627 row or as Table II A or IIB the 5th or 7 be listed as, the 251st, 255,259, its function homologue described in polypeptide or the literary composition shown in 261 and 627 row, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 251st, 255,259, nucleic acid molecule or its function homologue as herein described shown in 261 or 627 row) sequence of coded polypeptide, and have the activity that the Lignoceric acid level of giving described in the literary composition increases.

Have to give and improve each fine chemicals cerinic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:27297 as SEQ ID, 27298 or 27299,27300,27301,27302 or 27303,27304 or 27305,27306,27307,27308 or 27322,27323, shown in 27324 or Table IV the 7th row, the 251st, 252,253, the sequence of consensus sequence shown in 254 and 259 row or as Table II A or IIB the 5th or 7 be listed as, the 251st, 252,253, its function homologue described in polypeptide or the literary composition shown in 254 and 259 row, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 251st, 252,253, nucleic acid molecule or its function homologue as herein described shown in 254 and 259 row) sequence of coded polypeptide, and have the activity that the cerinic acid level of giving described in the literary composition increases.

Have to give and improve each fine chemicals myricyl acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:27311 as SEQ ID, 27312,27313 or 27314,27315,27316,27317,27318, shown in 27319 or Table IV the 7th row, the sequence of consensus sequence shown in the 256th and 257 row or as Table II A or IIB the 5th or 7 be listed as, the 256th, its function homologue described in polypeptide or the literary composition shown in 257 and 260 row, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 256th, nucleic acid molecule or its function homologue as herein described shown in 257 and 260 row) sequence of coded polypeptide, and have the activity that the myricyl acid level of giving described in the literary composition increases.

Have to give and improve each fine chemicals docosoic acid quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:27320 as SEQ ID, shown in 27321 or Table IV the 7th row, the sequence of consensus sequence shown in the 258th row or as Table II A or IIB the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in the 258th row, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in the 258th row) sequence of coded polypeptide, and have the activity that the docosoic acid level of giving described in the literary composition increases.

[0050.0.20.20] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, the sylvite of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid or sodium salt, perhaps their ester or glucoside (for example diglucoside).

[0051.0.20.20] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise the composition of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.

[0052.0.0.20] sees [0052.0.0.0]

[0053.0.20.20] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention (for example having shown in Table II A or IIB the 3rd row, 251-261,627 row the active polypeptide of protein or its homologue (for example Table II A or IIB the 5th or 7 row, 251-161 capable shown in)) and is expressed and increase, and has the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II A or IIB the 5th or 7 row, 251-261,627 row) shown in Table II A or IIB the 3rd row, 251-261,627 row) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses raising;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II A or IIB the 5th or 7 row, 251-261,627 row) shown in Table II A or IIB the 3rd row, 251-261,627 row), perhaps reduce the inhibition of polypeptide of the present invention regulated and control;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II A or IIB the 5th or 7 row, 251-261,627 row) shown in Table II A or IIB the 3rd row, 251-261,627 row) expression raising;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention " for example have protein or the active polypeptide of its homologue (for example shown in Table II A or IIB the 5th or 7 row, 251-261,627 row) shown in Table II A or IIB the 3rd row, 251-261,627 row) the expression raising;

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II A or IIB the 5th or 7 row, 251-261,627 row) shown in Table II A or IIB the 3rd row, 251-261,627 row) expression raising;

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have shown in Table II A or IIB the 3rd row, 251-261,627 row protein or its homologue (for example Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in) active polypeptide);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide (for example having protein or the active polypeptide of its homologue (shown in for example Table II A or IIB the 5th or 7 row, 251-261,627 go) shown in Table II A or IIB the 3rd row, 251-261,627 row).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.20.20] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improve coded protein or have shown in Table II A or IIB the 3rd or 5 row, 251-261,627 row protein or its homologue (polypeptide shown in for example Table II A or IIB the 5th or 7 row, 251-261,627 go) active expression of polypeptides or activity after give each fine chemicals increase.

[0055.0.0.20] to [0067.0.0.20]: see that [0055.0.0.0] is to [0067.0.0.0]

[0068.0.20.20] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.

[0069.0.0.20] sees [0069.0.0.0]

[0070.0.20.20] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example favourable docosoic acid, Lignoceric acid, the composition of cerinic acid and/or myricyl acid or their biological chemistry derivative for example contains high level (from the physiology of nutrition angle) docosoic acid, Lignoceric acid, the composition of cerinic acid and/or myricyl acid or their derivative.

[0071.0.0.20] sees [0071.0.0.0]

[0072.0.0.20]％

[0073.0.20.20] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of polypeptide of the present invention or its homologue (for example shown in Table II A or IIB the 5th or 7 row, 251-261,627 row) or, for example give each fine chemicals raising in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation.

[0074.0.20.20] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded.

[0075.0.0.20] to [0077.0.0.20]: see that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.20.20] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes.Method well known to those skilled in the art be can use and purified fermentation broth, tunning, plant or plant product come.For these different methods of setting up gradually, product is docosoic acid, Lignoceric acid, cerinic acid or myricyl acid or the composition that contains docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.20] to [0084.0.0.20]: see that [0079.0.0.0] is to [0084.0.0.0]

[0084.0.0.20]％

[0085.0.20.20] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th or 7 row, 251-261,627 row, perhaps

(b) genetic regulatory element that effectively is connected with nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th or 7 row, 251-261,627 row, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.20] to [0087.0.0.20]: see that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.20.20] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Stronger and output is enhanced to the resistance of biological and abiotic stress because the nutritive value that for example is used for the plant of raise poultry depends on above-mentioned fine chemicals or plant, so this is very important for the plant breeder.

[0088.1.0.20] sees [0088.1.0.0]

[0089.0.0.20] to [0090.0.0.20]: see that [0089.0.0.0] is to [0090.0.0.0]

[0091.0.0.20] sees [0091.0.0.0]

[0092.0.0.20] to [0094.0.0.20]: see that [0092.0.0.0] is to [0094.0.0.0]

[0095.0.20.20] advantageously increases docosoic acid, Lignoceric acid, cerinic acid or myricyl acid pond to separate a large amount of pure basically fine chemicals and/or to obtain the resistance of the raising of biology and abiotic stress and obtain high yield by method of the present invention in genetically modified organism.

[0096.0.20.20] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.

[0097.0.0.20]％

[0098.0.20.20] in preferred embodiments, each fine chemicals be produce according to the present invention and carry out isolating where necessary.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.20.20].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition that comprises each fine chemicals.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.20.20] comprises the transgenic plant that the method according to this invention synthetic comprises fine chemicals such as docosoic acid, Lignoceric acid, cerinic acid or myricyl acid and can advantageously directly put on market, and do not need institute's synthetic fine chemicals is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet each fine chemicals that the method according to this invention produces can also be as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free fine chemicals from biology, advantageously separate from plant.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because docosoic acid, Lignoceric acid, cerinic acid or myricyl acid are positioned at cell in the microorganism, must relate to separation to their recovery to biomass.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.

[0101.0.20.20] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 11 27-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.20.20] for example, docosoic acid, Lignoceric acid, cerinic acid or myricyl acid can separate and MS (mass spectrum) detection method is advantageously analyzed by HPLC, LC or GC.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect the existence of the product that contains docosoic acid, Lignoceric acid, cerinic acid or myricyl acid to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

[0103.0.20.20] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have the sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has the sequence shown in capable as Table I A or IB the 5th or 7 row, 251-261,627;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer of sequence shown in Table III the 7th row, 251-261,627 row obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise consensus sequence with sequence shown in Table IV the 7th row, 251-261,627 row and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding schedule IIA or IIB the 5th or 7 row, 251-261,627 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0104.0.20.20] in one embodiment, the sequence difference is one or more Nucleotide shown in nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 251-261,627 row.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method is not made up of sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row.In one embodiment, the identity of sequence shown in nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 251-261,627 row is less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule polypeptide of sequence shown in coding schedule IIA or IIB the 5th or 7 row, 251-261,627 row not.

[0105.0.0.20] to [0107.0.0.20]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.20.20] method of the present invention and had Table I A or IB the 5th or 7 row, 251-261, the nucleic acid molecule of sequence shown in 627 row, from Table II A or IIB the 5th or 7 row, 251-261, aminoacid sequence shown in 627 row is derived or is listed as from containing Table IV the 7th, 251-261, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 627 row, perhaps its coding has as Table II A or IIB the 3rd, 5 or 7 row, 251-261, the enzymic activity of polypeptide or bioactive polypeptide shown in 627 row or for example to give each fine chemicals behind its expression or active the increasing be docosoic acid, Lignoceric acid, derivative or homologue that cerinic acid or myricyl acid increase.

[0109.0.20.20] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make each fine chemicals, particularly docosoic acid, Lignoceric acid, cerinic acid or myricyl acid that the inventive method produced be optimized synthetic.

[0110.0.0.20] sees [0110.0.0.0]

[0111.0.0.20] sees [0111.0.0.0]

The nucleic acid molecule that uses in [0112.0.20.20] the inventive method is the isolated nucleic acid sequences form, its coding has the polypeptide of polypeptide active shown in Table II A or IIB the 3rd row, 251-261,627 row or has the polypeptide of peptide sequence shown in Table II A or IIB the 5th and 7 row, 251-261,627 row, and gives docosoic acid, Lignoceric acid, cerinic acid or the increase of myricyl acid level.

[0113.0.0.20] to [0114.0.0.20]: see that [0113.0.0.0] is to [0114.0.0.0]

[0115.0.0.20] sees [0115.0.0.0]

[0116.0.0.20] to [0120.0.0.20] sees that [0116.0.0.0] is to [0120.0.0.0]

[0120.1.0.20]：％

[0121.0.20.20] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II A or IIB the 5th or 7 be listed as, 251-261, peptide sequence or its functional homologue as herein described have the difference of one or more amino acid moleculars shown in 627 row, described artificial sequence is preferably given aforementioned activity, promptly improving Table II A or IIB the 5th or 7 row, the 251st, 255, giving the Lignoceric acid level after the activity of peptide sequence shown in 259 or 261 row increases, perhaps improving Table II A or IIB the 5th or 7 row, the 251st, 252,253, giving the cerinic acid level after the activity of peptide sequence shown in 254 or 259 row increases, perhaps improving Table II A or IIB the 5th or 7 row, the 256th, giving the myricyl acid level after the activity of peptide sequence shown in 257 or 260 row increases, and is perhaps improving Table II A or IIB the 5th or 7 row, giving the docosoic acid level after the activity of peptide sequence shown in the 258th row increases.

[0122.0.0.20] to [0127.0.0.20]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.20.20] polymerase chain reaction (PCR) amplification is used (for example the primer shown in Table III the 7th row, 251-261,627 row to) can be based on sequence shown in this paper, for example sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row or from as sequence deutero-sequence generation shown in Table II A or IIB the 5th or 7 row, 251-261,627 go.

[0129.0.20.20] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence is compared since then shown in Table IV the 7th row, 251-259,261 and 627 row.

[0130.0.20.20] can use then degenerated primer by pcr amplification have aforementioned activity (as improve to express or activity after give docosoic acid, Lignoceric acid, cerinic acid or myricyl acid and increase) the new protein fragment or comprise as described in segmental protein.

[0131.0.0.20] to [0138.0.0.20]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.20.20] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with for the Table I A of Lignoceric acid or IB the 5th or 7 row, the 251st, 255,259, sequence shown in 261 or 627 row or for the Table I A of cerinic acid or IB the 5th or 7 row, the 251st, 252,253, sequence shown in 254 or 259 row or for the Table I A of myricyl acid or IB the 5th or 7 row, the 256th, sequence shown in 257 or 260 row or for the Table I A of docosoic acid or IB the 5th or 7 row, sequence hybridization shown in the 258th row, and the coding expression has each fine chemicals, the i.e. docosoic acid of increasing, Lignoceric acid, the active peptide of cerinic acid or myricyl acid.

[0140.0.0.20] to [0146.0.0.20]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.20.20] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With nucleotide sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row (preferably, nucleic acid molecule of the present invention is the functional homologous compound of nucleic acid molecule shown in Table I B the 5th or 7 row, 251-261,627 row or identical with it) one of the complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.20.20] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I A or IB the 5th or 7 row, 251-261, nucleotide sequence or its portion homologous are at least about 30% shown in 627 row, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly having after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue increases docosoic acid, Lignoceric acid, the activity of cerinic acid or myricyl acid.

[0149.0.20.20] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I A or IB the 5th or 7 row, 251-261, nucleotide sequence (preferably shown in 627 row, nucleic acid molecule of the present invention is Table I B the 5th or 7 row, 251-261, or its part hybridization one of the functional homologous compound of nucleic acid molecule or identical shown in 627 row) with it, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and (as gives docosoic acid, Lignoceric acid, cerinic acid or myricyl acid increase) and randomly as Table II A or IIB the 5th row, 251-261, the protein of protein active shown in 627 row.

[00149.1.20.20] randomly, in one embodiment, with nucleotide sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row (preferably, nucleic acid molecule of the present invention is the functional homologous compound of nucleic acid molecule shown in Table I B the 5th or 7 row, 251-261,627 row or identical with it) one of the nucleotide sequence of hybridization have another or various active, described activity is for being known activity or being used for these protein of note as Table II A or IIB the 3rd row, 251-261,627 protein shown in capable.

[0150.0.20.20] in addition, nucleic acid molecule of the present invention can only contain Table I A or IB the 5th or 7 row, 251-261, nucleotide sequence (preferably shown in 627 row, nucleic acid molecule of the present invention is Table I B the 5th or 7 row, 251-261, the functional homologous compound of nucleic acid molecule or identical shown in 627 row) part of one of coding region with it, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give docosoic acid, Lignoceric acid, cerinic acid or myricyl acid increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and there be hybridizing at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides of mutant in described nucleotides sequence column region with the antisense sequences of one of sequence shown in sense strand, Table I A or the IB the 5th of one of sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row or 7 row, 251-261,627 row or its are natural under stringent condition.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With the right PCR of primer shown in Table III the 7th row, 251-261,627 row will produce as Table I A or IB the 5th or 7 row, 251-261,627 capable shown in fragment or its gene product of polynucleotide sequence.

[0151.0.0.20]: see [0151.0.0.0]

[0152.0.20.20] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and are listed as Table II A or IIB the 5th or 7,251-261, the abundant homology of aminoacid sequence shown in 627 row, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or raising Lignoceric acid the (the 251st as be shown in the examples, 255,259,261,627 go) or cerinic acid the (the 251st, 252,253,254,259 go) or myricyl acid the (the 256th, 257,260 row) or the activity of docosoic acid (the 258th row) level.

[0153.0.20.20] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, the amino-acid residue that described aminoacid sequence comprises with the aminoacid sequence minimal number is identical or of equal value shown in Table II A or IIB the 5th or 7 row, 251-261,627 row (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, shown in Table II A or IIB the 5th or 7 row, 251-261,627 row protein or its part have Table II A for example as herein described or IIB the 3rd row, 251-261,627 capable shown in the activity of polypeptide.

[0154.0.20.20] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein and complete amino acid sequence homology shown in Table II A or IIB the 5th or 7 row, 251-261,627 row be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% also most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.20] to [0156.0.0.20]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.20.20] the present invention relate in addition owing to the genetic code degeneracy be different from one of nucleotide sequence (with its part) shown in Table I A or IB the 5th or 7 row, 251-261,627 row and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase), for example comprise shown in Table IV the 7th row, 251-261,627 row polypeptide of sequence or as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in the nucleic acid molecule of polypeptide or its function homologue.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment shown in Table IV the 7th row, 251-259 and 261 row consensus sequence or as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise shown in Table IV the 7th row, 251-259 and 261,627 row consensus sequence or as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise the sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row.

[0158.0.0.20] to [0160.0.0.20]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.20.20] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (nucleic acid molecule that for example comprises sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.20] sees [0162.0.0.0]

[0163.0.20.20] preferably, under stringent condition with the nucleic acid molecule of the present invention of sequence hybridization shown in Table I A or IB the 5th or 7 row, 251-261,627 row corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals quantity increase in biology or its part (for example tissue, cell or cell chamber) after using activity of proteins).

[0164.0.0.20] sees [0164.0.0.0]

[0165.0.20.20] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row).

[0166.0.0.20] to [0167.0.0.20]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.20.20] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.The contained sequence of these amino acid sequence of polypeptide and sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row is different, but has kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with aminoacid sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II A or IIB the 5th or 7 be listed as, 251-261, sequence shown in 627 row is identical at least about 60%, more preferably with as Table II A or IIB the 5th or 7 be listed as, 251-261, one of sequence shown in 627 row is identical at least about 70%, even more preferably with as Table II A or IIB the 5th or 7 be listed as, 251-261, sequence shown in 627 row is at least about 80%, 90% or 95% homology, and most preferably with as Table II A or IIB the 5th or 7 be listed as, 251-261, sequence shown in 627 row is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.20] to [0172.0.0.20]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.20.20] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:25525 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:25525 sequence before use.

[0174.0.0.20]: see [0174.0.0.0]

[0175.0.20.20] for example, the sequence that has 80% homology at protein level and SEQ ID NO:25526 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:25526 sequence.

[0176.0.20.20] is by replacing, insert or lack from being listed as Table II A or IIB the 5th or 7 according to of the present invention, 251-261, the functional equivalent that one of polypeptide obtains shown in 627 row be listed as according to of the present invention as Table II A or IIB the 5th or 7,251-261, one of polypeptide has at least 30% shown in 627 row, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II A or IIB the 5th or 7 row, 251-261, polypeptide shown in 627 row has essentially identical character and discerns.

[0177.0.20.20] is by replacing, insert or lack from being listed as Table I A or IB the 5th or 7 according to of the present invention, 251-261, the functional equivalent that obtains of nucleotide sequence shown in 627 row be listed as according to of the present invention as Table II A or IIB the 5th or 7,251-261, one of polypeptide has at least 30% shown in 627 row, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II A or IIB the 5th or 7 be listed as, 251-261, the polypeptide of the essentially identical character of polypeptide shown in 627 row.

[0178.0.0.20] sees [0178.0.0.0]

[0179.0.20.20] can be by introducing replacement, interpolation or the disappearance of one or more Nucleotide in the nucleotide sequence of nucleic acid molecule of the present invention (particularly shown in Table I A or IB the 5th or 7 row, 251-261,627 row), and thereby replace, add or disappearance and producing is encoded as the nucleic acid molecule of the homologue of protein sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 go to introducing one or more amino acid in the coded protein.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to shown in Table I A or IB the 5th or 7 row, 251-261,627 row, introducing sudden change in the encoding sequence.

[0180.0.0.20] to [0183.0.0.20]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.20.20] is employed to have as Table I A or IB the 5th or 7 row, 251-261, the nucleotide sequence homologous compound of sequence shown in 627 row, perhaps come Table II A or IIB the 5th or 7 row freely, 251-261, the homologous compound of nucleotide sequence of sequence shown in 627 row also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from sequence shown in table LA or IB the 5th or 7 row, 251-261,627 row or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.20.20] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more sequences shown in Table I A or IB the 5th or 7 row, 251-261,627 row.In one embodiment, the preferred nucleic acid molecule comprises other Nucleotide the least possible, that do not show in the arbitrary sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method is identical with sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row.

The employed one or more nucleic acid molecule encodings of [0186.0.20.20] also preferred the inventive method comprise polypeptide of sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method is identical with sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row.

[0187.0.20.20] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises shown in Table II A or IIB the 5th or 7 row, 251-261,627 row polypeptide of sequence and contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in the encoding sequence of sequence identical.

The polypeptide (=protein) that [0188.0.20.20] still has the basic the enzyme activity (being its active basic reduction that do not have) of the polypeptide of the present invention of giving each fine chemicals increase is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, actively compare basic not reduction with polypeptide expressed activity shown in Table II A or IIB the 5th or 7 row, 251-261,627 row and under the same conditions.

[0189.0.20.20] homologous compound of sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of Table II A or IIB the 5th or 7 row, 251-261,627 sequence shown in capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.20]: see [0190.0.0.0]

[0191.0.20.20] compares with biological or its part of described contrast or selection in one embodiment, according to the inventive method described herein should biology or its part each fine chemicals level of producing protein bound improve.

[0192.0.0.20] to [0203.0.0.20]: see that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.20.20] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II A or IIB the 5th or 7 row, 251-261,627 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly Lignoceric acid (the 251st, 255,259,261,627 row) or cerinic acid (the 251st, 252,253,254,259 row) or myricyl acid (the 256th, 257,260 row) or docosoic acid (the 258th row) quantity increase;

(b) comprise, preferably comprise nucleic acid molecule or its segmental nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 251-261,627 row of mature form at least, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by use primer or the nucleic acid molecule of primer to increasing and obtain shown in Table III the 5th or 7 row, 251-261,627 row from cDNA library or genomic library, and in biological or its part, give each fine chemicals, i.e. the increase of Lignoceric acid (the 251st, 255,259,261,627 row) or cerinic acid (the 251st, 252,253,254,259 row) or myricyl acid (the 256th, 257,260 row) or docosoic acid (the 258th row) quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like consensus sequence shown in Table IV the 7th row, 252-259,261 and 627 row and give each fine chemicals in biology or its part, i.e. the nucleic acid molecule of Lignoceric acid (the 251st, 255,259,261,627 row) or cerinic acid (the 251st, 252,253,254,259 row) or myricyl acid (the 256th, 257,260 row) or docosoic acid (the 258th row) quantity increase;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be the nucleic acid molecule that Lignoceric acid (the 251st, 255,259,261,627 row) or cerinic acid (the 251st, 252,253,254,259 row) or myricyl acid (the 256th, 257,260 row) or docosoic acid (the 258th row) quantity increase, described peptide coding is as the structural domain of the polypeptide shown in Table II A or IIB the 5th or 7 row, 251-261,627 go; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I A or IB the 5th or 7,251-261, nucleic acid molecule shown in 627 row or coding (optimized encoding is mature form at least) are as Table II A or IIB the 5th or 7 row, 251-261, at least the 15nt of the nucleic acid molecule of polypeptide shown in 627 row, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from the sequence shown in capable as Table I A or IB the 5th or 7 row, 251-261,627 by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention is not made up of the sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row.In another embodiment, sequence at least 30% is identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99% shown in nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 251-261,627 row.In another embodiment, the nucleic acid molecule peptide sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row of not encoding.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 251-261,627 row at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in the protein of sequence.Therefore, in one embodiment, therefore, in one embodiment, (a) do not form to the coded protein of the nucleotide sequence of (l) by sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 251-261,627 row at least 30% identical and with Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.20] to [0206.0.0.20]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.20.20] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of glutamic acid metabolism, phosphoenolpyruvic acid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.20] to [0226.0.0.20]: see that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.20.20] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except the sequence or derivatives thereofs that Table I A or IB the 5th or 7 row, 251-261,627 row are mentioned, can advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, the extra polypeptide of expressing other genes of at least one acetyl-CoA or malonyl--CoA pathways metabolism or having very-long-chain fatty acid acid acyl (VLCFA) CoA synthase activity in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with one or more sequences shown in Table I A or IB the 5th or 7 row, 251-261,627 row with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.20.20]％

[0229.0.20.20] can be coding lipid acid approach (other gene of acetyl-CoA or malonyl--CoA) or have other gene of the polypeptide of very-long-chain fatty acid acyl (VLCFA) CoA synthase activity for example with employed other the favourable nucleotide sequences that sequence and/or the combination of aforementioned biosynthesis gene are expressed shown in Table I A or IB the 5th or 7 row, 251-261,627 row of present method.These genes cause the synthetic increase of VLCFA.

[0230.0.0.20] sees [230.0.0.0]

[0231.0.20.20] is in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded docosoic acid, Lignoceric acid, cerinic acid or myricyl acid simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.For example it be known to those skilled in the art that, to the inhibition of the enzyme of degraded docosoic acid, Lignoceric acid, cerinic acid or myricyl acid or check and to cause docosoic acid in the plant, Lignoceric acid, cerinic acid or myricyl acid accumulation to increase.

[0232.0.0.20] to [0276.0.0.20]: see that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.20.20] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.

[0278.0.0.20] to [0282.0.0.20]: see that [0278.0.0.0] is to [0282.0.0.0]

[0283.0.20.20] in addition, can from cell, separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II A or IIB the 3rd row, 251-261,627 capable shown in protein, the perhaps antibody of polypeptide or its antigen part shown in Table II A or IIB the 5th or 7 row, 251-261,627 row, it can utilize polypeptide of the present invention or its fragment to produce by standard technique.Monoclonal antibody preferably.

[0284.0.0.20] sees [0284.0.0.0]

[0285.0.20.20] in one embodiment, the present invention relates to have shown in Table II A or IIB the 5th or 7 row, 251-261,627 row sequence or by as Table I A or IB the 5th or 7 row, 251-261,627 capable shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.20.20] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in 251-259 and 261 and 627 row or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence or by its polypeptide of forming shown in 251-259 and 261 and 627 row, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to the polypeptide that comprises more than a consensus sequence (each row) shown in Table IV the 7th row, 251-259,261 and 627 row.

[0287.0.0.20] to [0289.0.0.20]: see that [0287.0.0.0] is to [0289.0.0.0]

[00290.0.20.20] uses Vector NTI Suite 8.0, (InforMax ^TM, Invitrogen ^TMLifescience software, U.S.Main Office, 7305 Executive Way, Frederick, MD21704, U.S.) assembly AlignX software (on September 25th, 2002) and use following setting to compare: for the pairing comparison: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Right for multiple ratio: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Breach separates the point penalty scope: 8; Residue is replaced matrix: blosum62; Hydrophilic residue: G P S N D Q E K R; Conversion weight: 0.5; Total calculation options: total residue mark: 0.9.Also selected to set in advance in order to compare conserved amino acid.

[0291.0.20.20] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 251-261, sequence shown in 627 row, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 251-261, sequence shown in 627 row.In another embodiment, described polypeptide of the present invention be can't help shown in Table II A or IIB the 5th or 7 row, 251-261,627 row sequence and is formed.

[0292.0.0.20] sees [0292.0.0.0]

[0293.0.20.20] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in other sequence of sequence phase region.In another embodiment, polypeptide of the present invention be can't help shown in Table II A or IIB the 5th or 7 row, 251-261,627 row sequence and is formed.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide is not made up of the coded sequence of nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 251-261,627 row.

[0294.0.20.20] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, 251-261, the polypeptide of activity of proteins shown in 627 row, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, 251-261, sequence shown in 627 row.

[0295.0.0.20] to [0296.0.0.20]: see that [0295.0.0.0] is to [0296.0.0.0]

[0297.0.0.20] sees [0297.0.0.0]

[00297.1.0.20]％

[0298.0.20.20] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with the abundant homologous aminoacid sequence of aminoacid sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used has and the identical aminoacid sequence of sequence shown in Table II A or IIB the 5th or 7 row, 251-261,627 row.

[0299.0.20.20] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I A or IB the 5th or 7 row, 251-261,627 capable shown in one of nucleotide sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises nucleotide sequence or the coded aminoacid sequence of its homologue with nucleotide sequence hybridization (preferably hybridize under stringent condition) shown in Table I A or IB the 5th or 7 row, 251-261,627 row.

[0300.0.20.20] is therefore, and be described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in sequence different.Therefore, this polypeptide contain with as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.20] sees [0301.0.0.0]

The biologically-active moiety of [0302.0.20.20] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II A or IIB the 5th or 7 row, 251-261, the aminoacid sequence shown in 627 row or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.20] sees [0303.0.0.0]

[0304.0.20.20] operation nucleic acid molecule of the present invention may cause generation have basically polypeptide shown in Table II A or IIB the 3rd row, 251-261,627 row activity and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.20] to [0308.0.0.20]: see that [0305.0.0.0] is to [0308.0.0.0]

[0309.0.20.20] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II A or IIB the 5th or 7 row, 251-261, protein shown in 627 row is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, and be not shown in Table II A or IIB the 5th or 7 row, 251-261, " other polypeptide " in 627 row is meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II A or IIB the 5th or 7 row, 251-261, polypeptide shown in 627 row is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II A or IIB the 3rd row, 251-261, proteinic and shown in 627 row from the protein of identical or different biology.In one embodiment, " other polypeptide " that are not shown in Table II A or IIB the 5th or 7 row, 251-261,627 row do not give that each fine chemicals increases in biology or its part.

[0310.0.0.20] to [0334.0.0.20]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.20.20] confirmed that the dsRNAi method is especially effectively also favourable to the expression that reduces nucleotide sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row and/or its homologue.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 go and/or its homologue.Double stranded rna molecule at the coded protein expression of the nucleotide sequence that is used for reducing shown in Table I A or IB the 5th or 7 row, 251-261,627 row sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.20] to [0342.0.0.20]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.20.20] as describing, in order to cause effective reduction of expression, at dsRNA and shown in Table I A or IB the 5th or 7 row, 251-261,627 row between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example the dsRNA that begins to produce from the sequence shown in Table I A or IB the 5th or 7 row, 251-261,627 row in a kind of organism or its homologue can be used for suppressing expressing accordingly in another organism.

[0344.0.0.20] to [0350.0.0.20]: see that [0344.0.0.0] is to [0350.0.0.0]

[0351.0.0.20] to [0361.0.0.20]: see that [0351.0.0.0] is to [0361.0.0.0]

[0362.0.20.20] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II A or IIB the 5th or 7 row, 251-261, polypeptide shown in 627 row, for example coding has protein such as Table II A or IIB the 3rd row, 251-261, the polypeptide of polypeptide active shown in 627 row) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Transgenosis with polypeptide of polypeptide active shown in Table II A or IIB the 5th or 7 row, 251-261,627 row is meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as polypeptide shown in Table II A or IIB the 3rd row, 251-261,627 row (for example have as Table II A or IIB the 5th or 7 row, 251-261,627 capable shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

[0363.0.0.20] sees [0363.0.0.0]

[0364.0.20.20] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---promotor of for example naturally occurring coding gene of polypeptide of the present invention shown in Table II A or IIB the 3rd row, 251-261,627 row with accordingly as Table I A or IB the 5th row, 251-261,627 capable shown in the combination of protein coding sequence---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.20] to [0373.0.0.20]: see that [0365.0.0.0] is to [0373.0.0.0]

[0374.0.20.20] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the docosoic acid that produces in the inventive method, Lignoceric acid, cerinic acid or myricyl acid, particularly each fine chemicals also can be from plant with free docosoic acid, Lignoceric acid, cerinic acid or each fine chemicals of myricyl acid, particularly free or be bonded to compound or the isolated in form of part (for example glucoside, for example diglucoside).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.20] to [0376.0.0.20]: see that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.20.20] therefore the invention still further relates to the method according to this invention, separated the docosoic acid that is produced, Lignoceric acid, cerinic acid or myricyl acid thus.

[0378.0.20.20] can separate by this way and be higher than 50% by weight, favourable is higher than 60%, preferably be higher than 70%, especially preferably be higher than 80%, extremely preferably be higher than 90% the docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid that produce in the method.As required, resultant docosoic acid, Lignoceric acid, cerinic acid or myricyl acid can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.20.20] is the mixture of each fine chemicals docosoic acid, Lignoceric acid, cerinic acid and/or myricyl acid by the product that the present invention produced in one embodiment.

The suitable synthetic parent material of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid that [0380.0.20.20] obtains by carrying out the inventive method as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises docosoic acid, Lignoceric acid, cerinic acid or myricyl acid composition that separation produces or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention be the inventive method docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid that produce or genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine purposes or be used to produce the purposes of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

[0381.0.0.20] to [0382.0.0.20]: see that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.20.20]

[0384.0.0.20] sees [0384.0.0.0]

The fermented liquid that [0385.0.20.20] obtains with this kind approach, particularly contain fermented liquid with other organic acid, amino acid, polypeptide or polysaccharide blended docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, usually dry matter content accounts for the 1-70% of weight, preferred 7.5 to 25%.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means and to utilize the concentration of sugar during this period of time to remain on or be reduced to 0-10g/l in the fermention medium, preferred to 0-3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.20.20] therefore can also be further purified the docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid that produce according to the present invention.For this reason, the composition that will contain product separates (by for example open column chromatography or HPLC), and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.20] to [0392.0.0.20]: see that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.20.20] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify and the nucleic acid molecule of the present invention nucleic acid molecule of under lax stringent condition, hybridizing of the nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 251-261,627 row particularly, and randomly separate full length cDNA clone or the complete genome group is cloned;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.20] to [0398.0.0.20]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.20.20] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving each fine chemicals generation raising under these conditions.

[00399.1.20.20] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect and whether depends on the active of polypeptide shown in Table II A or IIB the 5th or 7 row, 251-261,627 row or its homologue or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high biology phenotype more much at one as activity of proteins shown in Table II A or IIB the 5th or 7 row, 251-261,627 go.

[0400.0.0.20] to [0415.0.0.20]: see that [0400.0.0.0] is to [0415.0.0.0]

[0416.0.0.20] sees [0416.0.0.0]

[0417.0.20.20] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor to docosoic acid, Lignoceric acid, cerinic acid or myricyl acid biosynthetic pathway has resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block docosoic acid, Lignoceric acid, cerinic acid or myricyl acid synthetic inhibitor.

The example of this type of inhibitor is acetamide oxide, acetamide oxide or chlor(o)acetamide, and they suppress biosynthetic first step of VLCFA, condensation (Matthes, 2001 of acyl group-CoA and malonyl--CoA-β-ketoacyl-CoA; Schmalfuss, J., Matthes, B, and P.

Chloroacetamide mode of action.Abstr.Meeting Weed Science Society ofAmerica, Toronto, 40,117-118,2000).Therefore, the plant of excessive generation docosoic acid, Lignoceric acid, cerinic acid and/or myricyl acid can resist chlor(o)acetamide type weedicide.

[0418.0.0.20] to [0423.0.0.20]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.20.20] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other organic acids.Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.20] to [0434.0.0.0]: see that [0425.0.0.0] is to [0434.0.0.0]

[0435.0.20.20] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.20.20] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coliand Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.20.20] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, PGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.20.20] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.20.20] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.20.20] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad 50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.20] sees [0441.0.0.0]

[0442.0.0.20] sees [0442.0.0.0]

[0443.0.0.20] sees [0443.0.0.0]

[0444.0.20.20] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.20.20] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.20] to [0450.0.0.20]: see that [0446.0.0.0] is to [0450.0.0.0]

[0451.0.0.20] sees [0451.0.5.5]

[0452.0.0.20] to [0453.0.0.20]: see that [0452.0.0.0] is to [0453.0.0.0]

[0454.0.20.20] embodiment 8: the influence that analyzing nucleic acid molecules produces docosoic acid, Lignoceric acid, cerinic acid or myricyl acid

[0455.0.20.20] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example docosoic acid, Lignoceric acid, cerinic acid or myricyl acid) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being docosoic acid, Lignoceric acid, cerinic acid or myricyl acid) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

[0456.0.0.20]: see [0456.0.0.0]

[0457.0.20.20] embodiment 9: the purifying of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid

[0458.0.20.20] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS, GC-MS or TLC can clearly detect the existence of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid to the biology analysis of recombinating.

It is biological to use the following step to analyze, as the total amount that produces in the inventive method used yeast: by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000 W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 4022S1V diluter with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.20ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, Hewlett Packard 1100 HPLC have been used, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Technologies, can be from Ultra Scientific Inc.250 Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6 * 250mm[Waters, 34 Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

[0459.0.20.20] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.0.20] sees [0460.0.0.0]

[0461.0.20.20] embodiment 10: clone SEQ ID NO:25525,25617,26065,26455,26631,26659,26795,26837,26983,27031,27035 and 97565 is used for expressing plant

[0462.0.0.20] sees [0462.0.0.0]

[0463.0.20.20] passes through pcr amplification SEQ ID NO:25525,25617,26065,26455,26631,26659,26795,26837,26983,27031,27035 and 97565 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.0.20] to [0466.0.0.20]: see that [0464.0.0.0] is to [0466.0.0.0]

[0466.1.0.20] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.20.20] selects following primer sequence for genes of SEQ ID NO:25525:

I) forward primer (SEQ ID NO:25615)

atgaaacatc?tgcatcgatt?ctttag

Ii) reverse primer (SEQ ID NO:25616)

ttaaactgat?ggacgcaaac?gaacg

Select following primer sequence for genes of SEQ ID NO:25617:

I) forward primer (SEQ ID NO:26063)

atggaaaagg?gtactgttaa?gtg

Ii) reverse primer (SEQ ID NO:26064)

ttatgcgact?gccgcttcta?cttc

Select following primer sequence for genes of SEQ ID NO:26065:

I) forward primer (SEQ ID NO:26453)

atgattaacc?gtatccgcgt?agtc

Ii) reverse primer (SEQ ID NO:26454)

ttaaaatgtt?tcccagtttg?gatcttg

Select following primer sequence for genes of SEQ ID NO:26455:

I) forward primer (SEQ ID NO:26629)

atgagtcgtt?tagtcgtagt?atcta

Ii) reverse primer (SEQ ID NO:26630)

ttacgcaagc?tttggaaagg?tagc

Select following primer sequence for genes of SEQ ID NO:26631:

I) forward primer (SEQ ID NO:26657)

atggctgaat?ggagcggcga

Ii) reverse primer (SEQ ID NO:26658)

ttagtattcc?cacgtctccg?gg

Select following primer sequence for genes of SEQ ID NO:26659:

I) forward primer (SEQ ID NO:26793)

atggagacca?atttttcctt?cgact

Ii) reverse primer (SEQ ID NO:26794)

ctattgaaat?accggcttca?atattt

Select following primer sequence for genes of SEQ ID NO:26795:

I) forward primer (SEQ ID NO:26835)

atgaatagcg?taaaaagagt?aaagct

Ii) reverse primer (SEQ ID NO:26836)

ctaggccaaa?gacatcttag?cca

Select following primer sequence for genes of SEQ ID NO:26837:

I) forward primer (SEQ ID NO:26981)

atgacgagac?gtactactat?taatc

Ii) reverse primer (SEQ ID NO:26982)

tcacttgttt?attttcgata?aaatttcc

Select following primer sequence for genes of SEQ ID NO:26983:

I) forward primer (SEQ ID NO:27029)

atgtcgcctt?tgagaaagac?ggtt

Ii) reverse primer (SEQ ID NO:27030)

tcactcttcc?aggtttgagt?acg

Select following primer sequence for genes of SEQ ID NO:27031:

I) forward primer (SEQ ID NO:27033)

atggcggttg?cgatcaaaaa?gga

Ii) reverse primer (SEQ ID NO:27034)

tcaattgata?aatgtacttt?caatgatg

Select following primer sequence for genes of SEQ ID NO:27035:

I) forward primer (SEQ ID NO:27295)

atggctcggg?gtgacggaca?t

(a) reverse primer (SEQ ID NO:27296)

tcatgcttct?tttgcgtgat?gcaat

Select following primer sequence for genes of SEQ ID NO:97565:

I) forward primer (SEQ ID NO:97767)

atgatatgct?cacctcagaa?caaca

Ii) reverse primer (SEQ ID NO:97768)

ttaaaaacag?aggctttttc?ctctgc

[0468.0.20.20] to [0470.0.20.20]: see that [0468.0.0.0] is to [0470.0.0.0]

The PCR product that [0470.1.20.20] utilizes the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBIFermentas) phosphorylation to produce by Pfu Turbo archaeal dna polymerase, and the clone enters in the binary vector of treated mistake.

[0471.0.20.20] sees [0471.0.0.0]

[0471.1.20.20] uses Pfu Turbo archaeal dna polymerase benefit flat in the second step building-up reactions by the DNA end of the PCR product that the Herculase archaeal dna polymerase produces.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Utilize T4DNA polysaccharase and use standard to visit and make program (for example MBI Fermentas) phosphorylation PCR product, and be cloned in the carrier of handling.

[0472.0.20.20] to [0479.0.20.20]: see that [0472.0.0.0] is to [0479.0.0.0]

[0480.0.20.20] embodiment 11: express the generation of SEQ ID NO:25525,25617,26065,26455,26631,26659,26795,26837,26983,27031,27035 or 97565 transgenic plant

[0481.0.0.20] to [0513.0.0.20]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.20.20] alternatively, as Beerthuis, R.K., Keppler, J.G., Nature.1957Apr 6; 179 (4562): detect Lignoceric acid, cerinic acid, myricyl acid or docosoic acid described in the 731-2.

The different plants of being analyzed the results are shown in following table 1:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??b0019	Cerinic acid (C26:0)	??GC	??1.40	??2.50
??b0019	Lignoceric acid (C24:0)	??GC	??1.46	??3.24
					??b0880	Cerinic acid (C26:0)	??GC	??2.72	??3.94
??b1886	Cerinic acid (C26:0)	??GC	??1.39	??4.35
					??b1896	Cerinic acid (C26:0)	??GC	??1.39	??1.75
??b3938	Lignoceric acid (C24:0)	??GC	??1.37	??1.89
					??YDR513W	Myricyl acid (C30:0)	??GC	??1.31	??2.08
??YER156C	Myricyl acid (C30:0)	??GC	??1.33	??1.73

??ORF	Metabolite	Method	??Min	??Max
					??YGL205W	Docosoic acid (C22:0)	??GC	??1.74	??2.32
??YHR201C	Cerinic acid (C26:0)	??GC	??1.46	??3.27
					??YHR201C	Lignoceric acid (C24:0)	??GC	??1.29	??3.71
??YLR255C	Myricyl acid (C30:0)	??GC	??1.34	??1.88
					??YPR138C	Lignoceric acid (C24:0)	??GC	??1.92	??2.60
??b0255	Lignoceric acid (C24:0)	??GC	??1.35	??2.15

[0515.0.20.20] the 2nd row have shown metabolite docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid of being analyzed.The 4th row and the 5th row have shown the ratio of metabolite between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

[0516.0.0.20] to [0552.0.0.20]: see that [0516.0.0.0] is to [0552.0.0.0]

[0552.1.0.20]/

[0552.2.0.20] embodiment 16: prepare homologous sequence from plant

Grow under the standard that different plants can be in the greenhouse or the condition of change.RNA can extract according to Jones, Dunsmuir and Bedbrook (1985) EMBO method J.4:2411-2418.The organization material of about 1 gram from Different Organs ground in liquid nitrogen.With powder transfer to containing 4.5ml NTES damping fluid (100mM NaCl, 10mM Tris/HCl pH 7.5,1mMEDTA, 1%SDS; Be dissolved in the water of no RNA enzyme) and the 13ml Falcon pipe of 3ml phenol/chloroform/primary isoamyl alcohol (25/24/1) in, mixing and being stored on ice immediately.Use whizzer (Sorval; SM24 or SS34 rotor) with 7000 rev/mins with centrifugal 10 minutes of mixture.Supernatant is transferred in the new pipe, adds the 3M sodium-acetate (pH 5.2, are dissolved in the water of no RNA enzyme) of 1/10 volume and the Virahol of 1 times of volume, mixes the back and stores 1 hour or spend the night in-20 ℃.With 7000 rev/mins of centrifugal mixtures 10 minutes.Abandoning supernatant is also with 70% ethanol (v/v) washing precipitation.With 7000 rev/mins of centrifugal mixtures 5 minutes, abandoning supernatant also will precipitate dry air.Adding 1ml does not have the water of RNA enzyme and makes DNA/RNA be deposited in 4 ℃ of dissolvings on ice.Be transferred to nucleic acid solution in the 2ml Eppendorf pipe and add 1ml 4M Lithium Acetate.Solution can or spend the night in 4 ℃ of placements 3 hours at least behind the mixing.With 14000 rev/mins of centrifugal mixtures 10 minutes, abandoning supernatant also precipitated with 70% washing with alcohol, and dry air also is dissolved in 200 μ l and does not have in the water of RNA enzyme.

Total RNA can be used for method (for example using the synthetic and clone's test kit of ZAP-cDNA of Stratagene, La Jolla, the U.S.) the construction cDNA library according to manufacturers.Basically, use oligo (dT) joint primer to start the synthetic article one chain of messenger RNA(mRNA) (mRNA), and use reversed transcriptive enzyme to carry out reverse transcription.After the second chain cDNA is synthetic, double-stranded cDNA is connected into Uni-ZAP XR carrier.Uni-ZAP XR carrier allows pBluescript phagocytosis plastochondria inscribe to remove.The polylinker of pBluescript phagemid has 21 unique cloning sites, and its both sides are T3 and T7 promotor, and can select to be used for dna sequencing to 6 different primer sites.The systemic single operation order-checking terminal to 5 expection initiations of clone can allow for example by means of pedant pro software package (Biomax, M ü nchen) sequence to be carried out preliminary note.Retrieve the clone that can identify nucleic acid of the present invention or be used for the nucleic acid of the inventive method based on the homology of using canonical algorithm (as blastp or gap).The supposition full-length clone with identity or high homology that is identified is further checked order, so that obtain full sequence.

As mentioned above, by prepare each cDNA library from different plant origins, can identify the homologous sequence that other is new in a similar manner.Can use available sequences of the present invention under low stringency condition, to screen the library then, for example as Sambrook etc., described in the Molecular Cloning:Alaboratory manual, Cold Spring Harbor 1989, Cold Spring HarborLaboratory Press.The positive colony of purifying can be used for cutting technique and sufficient sequence analysis in the body.Use blastp or gap program can identify directly that to the paired sequence alignment of original series and new sequence to homologue (ortholog) (meaning is the homologous sequences from different biologies), it should have at least 30% sequence identity.In addition, the conservative property of function important amino acid residue or structural domain (can identify by comparing several paralogs that obtained) can be identified as new straight new sequence to homologue.

In addition, the library can be used for extensive sequencing and the sequence that obtained can be stored in the sequence library, can screen this sequence library by different searching algorithm (for example utilizing the tbastn algorithm of the nucleotide sequence that aminoacid sequence search of the present invention obtained) then and seek supposition directly to homologue.Clone with highest serial identity is used for full sequence and determines and directly can identify as mentioned above to homologue.

[0553.0.20.20]

1. produce the method for docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, it comprises:

(a) improve in non-human being or its one or more parts or produce as Table II A or IIB the 5th or 7 row, 251-261,627,627 capable shown in the activity of protein or its function equivalent; With

(b) in allowing described biology, produce under the condition of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid and cultivate this biology.

2. produce the method for docosoic acid, Lignoceric acid, cerinic acid or myricyl acid, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, 251-261,627,627 row, and described nucleic acid molecule is given docosoic acid in biology or its part, Lignoceric acid, cerinic acid or the increase of myricyl acid quantity;

B) contain nucleic acid molecule just like the nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 251-261,627,627 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that docosoic acid in biology or its part, Lignoceric acid, cerinic acid or myricyl acid quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 251-261,627,627 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives docosoic acid in biology or its part, Lignoceric acid, cerinic acid or the increase of myricyl acid quantity;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give docosoic acid in biology or its part, Lignoceric acid, cerinic acid or myricyl acid quantity and increase;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 251-259 and 261,627 row and give biology or its part in docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give docosoic acid in biology or its part, Lignoceric acid, cerinic acid or the increase of myricyl acid quantity, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or bonded docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, 251-261,627,627 row, and described nucleic acid molecule is given docosoic acid in biology or its part, Lignoceric acid, cerinic acid or the increase of myricyl acid quantity;

B) contain nucleic acid molecule just like the nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 251-261,627,627 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that docosoic acid in biology or its part, Lignoceric acid, cerinic acid or myricyl acid quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 251-261,627,627 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives docosoic acid in biology or its part, Lignoceric acid, cerinic acid or the increase of myricyl acid quantity;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give docosoic acid in biology or its part, Lignoceric acid, cerinic acid or myricyl acid quantity and increase;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 251-259 and 261,627 row and give biology or its part in docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give docosoic acid in biology or its part, Lignoceric acid, cerinic acid or the increase of myricyl acid quantity, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt; Thus, nucleic acid molecule is different from sequence shown in Table I A or IB the 5th or 7 row, 251-261,627,627 row by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 251-261,627,627 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps docosoic acid, Lignoceric acid, cerinic acid or myricyl acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid level that will measure or expression of polypeptides level and standard docosoic acid, Lignoceric acid, cerinic acid or myricyl acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and give the method that docosoic acid in plant or the microorganism, Lignoceric acid, cerinic acid or myricyl acid produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in docosoic acid, Lignoceric acid, the polypeptide that cerinic acid or myricyl acid quantity increase and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and it is coded and give docosoic acid in biology or its part to be provided at the nucleic acid molecule of described read-out system of permission and claim 6, Lignoceric acid, response compound and described polypeptide bonded detectable signal under the condition of the expression of polypeptides that cerinic acid or myricyl acid quantity increase; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and give the method that docosoic acid in the cell, Lignoceric acid, cerinic acid or myricyl acid produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that docosoic acid, Lignoceric acid, cerinic acid or myricyl acid increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce docosoic acid, Lignoceric acid, cerinic acid or myricyl acid;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give docosoic acid in the host cell, Lignoceric acid, cerinic acid or myricyl acid level after it is expressed and improve with wild-type.

20. identify and give the method that docosoic acid in the cell, Lignoceric acid, cerinic acid or myricyl acid produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that docosoic acid, Lignoceric acid, cerinic acid or myricyl acid quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce docosoic acid, Lignoceric acid, cerinic acid or myricyl acid;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) docosoic acid, Lignoceric acid, cerinic acid or the myricyl acid level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give docosoic acid in the host cell, Lignoceric acid, cerinic acid or myricyl acid level after it is expressed and improve with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that docosoic acid, Lignoceric acid, cerinic acid or myricyl acid increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control docosoic acid, Lignoceric acid, cerinic acid or myricyl acid level at biology.

25. agrochemicals, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of any one described method of claim 1-5, claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein fine chemicals is docosoic acid, Lignoceric acid, cerinic acid or myricyl acid.

27. according to claim 10-12 any one described host cell or plant, its anti-docosoic acid, Lignoceric acid, cerinic acid or biosynthetic weedicide of myricyl acid of suppressing.

[0554.0.0.20] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.21] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.21] sees [0001.0.0.0] for disclosing of this paragraph.

[0002.0.21.21] plant produces glycerine and glycerol-3-phosphate.Importantly, be eukaryotic main component by the glycerine lipid that forms of deriving.From dry weight, in any case they all can account for the cell total mass 10% to 90% between.Triacylglycerol is the main source of energy storage in the eukaryote.

Glycerol-3-phosphate can be synthetic by two kinds of different routes in plant.In a route,, form glycerol-3-phosphate from phosphodihydroxyacetone (DHAP) (glucolytic intermediate product) by the serial action of triosephosphate isomerase, phosphoglyceraldehyde Phosphoric acid esterase, Glycerose reductase enzyme and glycerol kinase.Last enzyme has been proposed the rate-limiting step as this glycerol-3-phosphate synthetic route in this approach.Glycerol-3-phosphate dehydrogenase in second approach (NAD (+)-G-3-P oxydo-reductase, EC 1.1.1.8) (GPDH) catalysis di(2-ethylhexyl)phosphate pyruvic alcohol (DHAP) reduction forms glycerol-3-phosphate (G-3-P).Based on the research of zymetology shown the activity of this enzyme in rape flower (Brassicacampestris) seed might be at least glycerol-3-phosphate main source (Sharma etc., 2001, Plant Sci 160,603-610).

[0003.0.0.21] has two types GPDH at least in plant, a kind of tenuigenin that is present in, and a kind of plastid that is present in, and they also are being different aspect its reduction cosubstrate.Kytoplasm GPDH utilizes NADH as cosubstrate.As before in other eukaryote, determining, the plastosome FAD dependency glycerol-3-phosphate dehydrogenase (FAD-GPDH) that mouse ear mustard belongs to forms G-3-P and shuttles back and forth, and in plant, kytoplasm G-3-P metabolism and utilization of carbon source and energy metabolism are connected-also see Shen W. etc., FEBS Lett.2003 February 11; 536 (1-3): 92-6.

The seedling that the insensitive mouse ear of glycerine mustard belongs to mutant: gli1 lacks glycerol kinase, accumulates glycerine and abiotic stress is had stronger resistance, sees Eastmond P.J., The Plant Journal, 2004,37 (4), 617-625.These data show that glycerine katabolism needs glycerol kinase in the mouse ear mustard genus, and show that the accumulation of glycerine can strengthen resistance multiple and the relevant abiotic stress that dewaters.

The main depot lipid (or oils) of [0004.0.0.21] seed exists with triacylglycerol (TAG) form, and perhaps three lipid acid are connected to glycerine by ester bond.Triacylglycerol is synthetic to relate to different cell chambers, comprises tenuigenin, plastosome, plastid and endoplasmic reticulum (ER).Glycerol-3-phosphate enters ER and carries out the final step of triacylglycerol synthetic.Recently the triacylglycerol of Xing Chenging accumulation between ER duplicature two-layer forms the oil by single (or half) unit membrane parcel.

[0005.0.0.21] glycerol-3-phosphate acyltransferase (GPAT) is one of most important enzyme in the TAG biosynthesizing, and initial TAG is synthetic because this enzyme is by the acylation generation Sn-1-acyl group-glycerol-3-phosphate of catalysis Sn-glycerol-3-phosphate Sn-1 position.Produce phosphatidic acid (PA) by LPA acyltransferase acetylize Ultrapole L (LPA) then.By the PA phosphohydrolase PA dephosphorylation is discharged diacylglycerol (DAG) then.At last, the activity by the DAG acyltransferase makes DAG become acylate.In the second approach, form phosphatidylcholine (PC), and the further desaturation of its acyl residue.Discharge the phosphorylcholine residue by hydrolytic action and corresponding D AG acetylize then.This second approach usually carries out in highly unsaturated TAG synthetic that (Heldt 1997, Plant biochemistry and molecular biology.The Oxford University Press, NewYork).In addition, present many researchs have proved that GPAT is relevant with plant cold resistance, see LiuJi-Mei etc., Plant Physiol.120 (1999): 934.

Glycerol-3-phosphate is the elementary substrate of triacylglycerol synthetic.(Planta 219 (2004): 827-835) shown to the seed glycerol injection of growing to cause the glycerol-3-phosphate level to improve for Vigeolas and Geigenberger.The glycerol-3-phosphate level improves and to be accompanied by sucrose and to increase to the flow of total lipid and triacylglycerol, and this just provides evidence to prove that high-caliber glycerol-3-phosphate limits the generation of triacylglycerol in the seed of growth equally.

Shown and pass through glycerine in sarcoplast and liver cell: the acetyl-CoA acyltransferase forms monoacylglycerol to the direct acylations effect of glycerine, and forms diacylglycerol and triacylglycerol (J.Lipid res.42 (2001) such as Lee D.P.: 1979-1986) subsequently.When the glycerol-3-phosphate approach weakens or when the glycerine level improved, this direct acylations effect became more outstanding.

In glycerinated water/ethanol plants extract and plant fragrance hydrocarbon (phytoaromatic) compound, glycerine uses with water and ethanol (ethyl hexanol) [0006.0.0.21].These products are as foodstuff additive, the enriched material of mineral substance, trace element, activeconstituents (alkaloids, polyphenol, pigment or the like) and the aromatic essence that is provided in the plant to be found.Glycerine can be used as the carrier of plant milk extract.Find that in end product (fresh plant extract) glycerol concentration is up to 24% or 25%.

Raw glycerine is that rapeseed oil forms vegetable seed methyl ester (RME) and used edible oil forms the byproduct that produces in the transesterification mechanism of used edible methyl ester (AME) (RME and AME are well-known biofuel)

Glycerine world production amount is estimated as about 750.000 tons/year.About 90% is based on natural oils and fat is processed.

[0007.0.0.21] for example set up the commercial source of large scale culturing Dunaliella salina (Dunaliella) green alga as glycerine recently.Dunaliella salina can tolerate high salt concn and keep the low interior salt concn of cell.Reach the level realization osmoregulation that the external penetration that contends with is pressed by accumulation glycerine in the cell.

The adjusting of phosphodihydroxyacetone (DHAP) reductase enzyme infiltration isoform (Osm-DHAPR) is the enzyme that has of Dunaliella tertiolecta only, and it is to participate in free glycerol to synthesize so that carry out the adjusting infiltration isoform of osmoregulation in extreme environment (for example high salinity), see Ghoshal D. etc., Protein Expression and Purification, 2002,24, (3), 404-411.

An open question is to understand glycerol-3-phosphate synthetic metabolism adjusting and modifying glyceryl ester metabolism or the aborning purposes of glycerine in plant biochemistry.In fact, reasonably genetically engineered plant for improve the synthetic of triacylglycerol or other valuable additional product and the glycerine synthesis capability imported important economic plants so as to improve the environmental stress tolerance will be very significant-see: Durba G. etc., J.Plant Biochemistry; Biotechnology 10 (2001), 113-120.

A kind of method that [0008.0.21.21] improves biosynthesizing throughput is to use recombinant DNA technology.Therefore, people are desirably in and produce glycerine and/or glycerol-3-phosphate in the plant.The inhuman production of this type allows the most suitable and the most effective quality, quantity and the screening of producing biology of control.The latter is particularly important for commercial production economics, and also is favourable for the human consumer therefore.In addition, people are desirably in the plant and produce glycerine and/or glycerol-3-phosphate, so that improve plant production power and to the resistance of biological and abiotic stress as mentioned above.

More already used year of recombinant DNA technology method is with by increasing different biosynthesis genes and study the production that its influence to fine chemicals production improves fine chemicals in microorganism and the plant.For example, it is reported that the xenthophylls astaxanthin can result from the nectary of rotaring gene tobacco plant.Those transgenic plant are to prepare by the carrier transformation of tobacco plant that agrobacterium tumefaciens mediates, and wherein carrier comprises the ketolase encoding gene (called after crtO) from H.pluvialis, and the Pds gene of tomato is as the promotor and the leader sequence of encoding.Those results show that about 75% carotenoid that is found in the conversion plant flowers contains ketone group.

[0009.0.21.21] if can obtain producing algae, plant or other microorganism of a large amount of glycerine and/or glycerol-3-phosphate, should be favourable therefore.The protokaryon or the eukaryotic microorganisms that relate to this type of conversion in some embodiments that the present invention is hereinafter discussed.

If it also should be favourable can obtaining root, leaf, stem, fruit or spend the middle plant that produces a large amount of glycerine and/or glycerol-3-phosphate.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

In addition, also should be favourable if can obtain producing in the seed a large amount of plants of lipids always.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

The quality that [0010.0.21.21] therefore improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the glycerine and/or the glycerol-3-phosphate that for example are present in as mentioned above in plant and some microorganisms are limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is specific glycerine and/or glycerol-3-phosphate spectrum in the balanced diet as far as possible, because excess of glycerol and/or glycerol-3-phosphate have negative effect on the specific concentrations.Further improving the quality only may be by adding other glycerine and/or glycerol-3-phosphate, and these compositions all are limited.

[0011.0.21.21] is necessary with balance mode glycerine and/or glycerol-3-phosphate to be added into suitable biology in order to guarantee the high quality of food and animal-feed.

[0012.0.21.21] therefore, still be starved of one or more codings and participate in glycerine and/or the biosynthetic enzyme of glycerol-3-phosphate or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but forever need to improve the method that obtains the highest intrinsic energy glycerine and/or glycerol-3-phosphate; On the other hand, reduce the byproduct that produces in the production process as far as possible.

In addition, still be starved of one or more codings and participate in the biosynthetic enzyme of total lipid or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but need forever to improve the method that obtains the total lipid of the highest intrinsic energy, on the other hand, reduce the byproduct that produces in the production process as far as possible.

Glycerine or glycerol-3-phosphate are the biosynthetic biosynthesizing precursors of monoacylglycerol, diacylglycerol, triacylglycerol, glycerine Serine and other glyceride (for example glycoglyceride, diphosphatidylglycerol, phosphoric acid fat, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositols, phytoglycolipid).Therefore, after total lipid extraction and the hydrolysis of lipid effect in pair cell, tissue or plant part such as seed and the leaf analysis of glycerol content directly relevant with analysis to total lipid content.For example, cause total lipid content raising in seed or the leaf if cross the biosynthetic gene of expression participation triacylglycerol in seed, this seed also will show the glycerol content raising after total lipid extraction and hydrolysis of lipid effect so.

Therefore, the method for the method that glycerine increases in the lipid part that causes after the fracture of ester functional group as described below as using methyl alcohol and hydrochloric acid mixture obviously to represent to improve triacylglycerol or total lipid to produce.

[0013.0.0.21] sees [0013.0.0.0] for disclosing of this section.

[0014.0.21.21] therefore in first embodiment, the present invention relates to produce the method for fine chemicals, and wherein fine chemicals is glycerine and/or glycerol-3-phosphate.Therefore, in the present invention, term " fine chemicals " is meant glycerine and/or glycerol-3-phosphate as used herein.In addition, term " fine chemicals " also refers to comprise the fine chemicals of glycerine and/or glycerol-3-phosphate as used herein.

[0015.0.21.21] in one embodiment, term " fine chemicals " meaning is meant glycerine.In one embodiment, depend on the context that uses it, term " fine chemicals " meaning is meant glycerol-3-phosphate.In whole specification sheets, term " fine chemicals " meaning is meant glycerine and/or glycerol-3-phosphate, its salt, ester, thioesters or free form or is bonded to other compound such as sugar or glycopolymers (for example glucoside or polyvalent alcohol such as inositol) form.

In one embodiment, term " fine chemicals " meaning be meant monoacylglycerol, diacylglycerol, triacylglycerol, glycerine Serine and/or other glyceride (such as but not limited to glycoglyceride, two glycerine Serines, phosphono fat, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositols or phytoglycolipid) and below be called " total fat ".

[0016.0.21.21] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YDR065W, b2441, b3457, YBR084W, YDR513W, YGL237C, YIL150C, YLR082C, YLR224W, YLR255C, YMR015C, YOR344C, YPL099C, YPL268W, b3644, YHR072W, b2710, b3498 or b4073 protein; With

(b) in allowing described biology, produce under the condition that fine chemicals is glycerine and/or glycerol-3-phosphate and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II the 3rd row, the 173rd, 262-274 and 628-632 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce under the condition that fine chemicals is glycerine and/or glycerol-3-phosphate and make biological growth.

[0016.1.21.21] therefore, term " fine chemicals " meaning is meant capable with Table I 262-274 and relevant " glycerine " or its homologue of the 628th and 629 listed full sequences of row, and the meaning is meant and Table I the 173rd row " glycerol-3-phosphate " or its homologue relevant with the capable listed sequence of 630-632.In addition, term " fine chemicals " meaning is meant " total fat ", and it includes but not limited to " monoacylglycerol, diacylglycerol, triacylglycerol, glycerine Serine and/or other glyceride (for example glycoglyceride, two glycerine Serines, phosphono fat, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositols or phytoglycolipid) " or its homologue relevant with Table I the 265th, 266,267,269,271,272,263 or the capable listed full sequence of 628-632.Therefore, term " fine chemicals " can refer to " glycerine ", " glycerol-3-phosphate " or " total fat " according to environment and context.

Above [0017.0.0.21] and [0018.0.0.21] sees for disclosing of [0017.0.0.21] and [0018.0.0.21] these paragraphs [0017.0.0.0] and [0018.0.0.0]

The method that [0019.0.21.21] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, the 173rd, protein active shown in 262-274 and 628-632 are capable or by as Table I the 5th or 7 row, the 173rd, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 262-274 and 628-632 were capable.

[0020.0.21.21] is surprisingly found out that, e. coli k12 protein b2441, b3457, b3644, b2710, b3498 or b4073 or yeast saccharomyces cerevisiae protein YDR065W, YBR084W, YDR513W, YGL237C, YIL150C, YLR082C, YLR224W, YLR255C, YMR015C, YOR344C, YPL099C, YPL268W or YHR072W protein transgene expression in Arabidopis thaliana give the glycerine of the plant that transforms and/or glycerol-3-phosphate and/or total fat (with regard to described protein and homologue thereof and coding nucleic acid molecule (particularly as Table II the 3rd be listed as, the 173rd, shown in 262-274 and 628-632 are capable), be " fine chemicals " or " each fine chemicals ") the content increase.

[0021.0.0.21] sees [0021.0.0.0] for disclosing of this paragraph

The sequence of [0022.0.21.21] yeast saccharomyces cerevisiae YDR065W has been published in Jacq, C. etc., and Nature 387 (6632 supplementary issue), 75-78 (1997), and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YDR065W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerol-3-phosphate, the preferred quantity of the glycerol-3-phosphate of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YDR065W is enhanced.

The sequence of e. coli k12 b2441 has been published in Blattner, F.R. etc., Science277 (5331), 1453-1474 (1997), and its activity to be defined as be the protein with thanomin deaminase active.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b2441 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b2441 is enhanced.

The sequence of e. coli k12 b3457 has been published in Blattner, F.R. etc., Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be the protein with branched-chain amino acid transport activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b3457 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine and/or total fat, preferably free or the glycerine of combining form and/or the quantity of total fat.In one embodiment, in the methods of the invention, the activity of protein b3457 is enhanced.

The sequence of yeast saccharomyces cerevisiae YBR084W has been published in Goffeau, A. etc., Science 274 (5287), 546-547 (1996), and its activity to be defined as be the protein with C1-tetrahydrofolic acid (THFA) synthase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YBR084W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YBR084W is enhanced.

The sequence of yeast saccharomyces cerevisiae YDR513W has been published in Jacq, C. etc., Nature 387 (6632 supplementary issue), 75-78 (1997), and its activity to be defined as be the protein with glutathione reductase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YDR513W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine and/or total fat, preferably free or the glycerine of combining form and/or the quantity of total fat.In one embodiment, in the methods of the invention, the activity of protein YDR513W is enhanced.

The sequence of yeast saccharomyces cerevisiae YGL237C has been published in Tettelin, Nature such as H. 387 (6632 supplementary issue), and 81-84 (1997), and its activity is defined as transcriptional activation agent and overall adjustment thing that vrg genes is expressed.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YGL237C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine and/or total fat, preferably free or the glycerine of combining form and/or the quantity of total fat.In one embodiment, in the methods of the invention, the activity of protein YGL237C is enhanced.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Churcher, C. etc., Nature 387 (6632 supplementary issue), 84-87 (1997), and its activity to be defined as be to have chromatin in conjunction with active protein.Therefore, in one embodiment, the inventive method comprises the protein YIL150C from yeast saccharomyces cerevisiae as shown here, for example the S phase is initial or finish the purposes of required protein or its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine and/or total fat, preferably free or the glycerine of combining form and/or the quantity of total fat.In one embodiment, in the methods of the invention, the activity of protein YIL150C is enhanced.

The sequence of yeast saccharomyces cerevisiae YLR082C has been published in Johnston, M. etc., Nature 387 (6632 supplementary issue), 87-90 (1997), and its activity to be characterized as being be the invalid lethal arrestin of Rad53.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YLR082C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YLR082C is enhanced.

The sequence of yeast saccharomyces cerevisiae YLR224W has been published in Johnston, M. etc., and Nature 387 (6632 supplementary issue), 87-90 (1997), and its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YLR224W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine and/or total fat, preferably free or the glycerine of combining form and/or the quantity of total fat.In one embodiment, in the methods of the invention, the activity of protein YLR224W is enhanced.

The sequence of yeast saccharomyces cerevisiae YLR255C has been published in EMBL Data Library, February nineteen ninety-five.MIPS_Reference:S59386, accession number: S69301, its activity is not still characterized.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YLR255C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YLR255C is enhanced.

Yeast saccharomyces cerevisiae YMR015C has been published in Bowman, S. etc., Nature 387 (6632 supplementary issue), 90-93 (1997), and its activity to be defined as be to have the active protein of C-22 sterol desaturase.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YMR015C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine and/or total fat, preferably free or the glycerine of combining form and/or the quantity of total fat.In one embodiment, in the methods of the invention, the activity of protein YMR015C is enhanced.

The sequence of yeast saccharomyces cerevisiae YOR344C has been published in Dujon, B. etc., Nature 387 (6632 supplementary issue), 98-102 (1997), and its activity to be defined as be the protein that is rich in Serine of involved in sugar glycolysis genetic expression.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YOR344C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine and/or total fat, preferably free or the glycerine of combining form and/or the quantity of total fat.In one embodiment, in the methods of the invention, the activity of protein YOR344C is enhanced.

The sequence of yeast saccharomyces cerevisiae YPL099C has been published in Bussey, H. etc., Nature 387 (6632 supplementary issue), 103-105 (1997), and its activity be defined as be the supposition membrane protein.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YPL099C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, the YPL099C activity of proteins is enhanced.

The sequence of yeast saccharomyces cerevisiae YPL268W has been published in Bussey, H. etc., Nature 387 (6632 supplementary issue), 103-105 (1997), and its activity to be defined as be the protein with phosphoinositide phospholipase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YPL268W of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, the YPL268W activity of proteins is enhanced.

The sequence of e. coli k12 b2710 (accession number NP_417190) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be flavine rubredoxin (FIRd) bi-functional NO and O ₂Reductase enzyme.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary flavine rubredoxin (FIRd) bi-functional NO and O ₂The purposes of reductase enzyme protein matter or its homologue, it is used for producing fine chemicals in biological or its part as described, be glycerol-3-phosphate and/or the glyceryl ester that comprises glycerol-3-phosphate, lipid, oils and/or fat, particularly increase glycerol-3-phosphate, the preferred quantity of the glycerol-3-phosphate of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b2710 is enhanced.In one embodiment, in the methods of the invention, flavine rubredoxin (FIRd) bi-functional NO and O ₂The activity of reductase enzyme protein matter is enhanced or produces, for example from colibacillary flavine rubredoxin (FIRd) bi-functional NO and O ₂The activity of reductase enzyme or its homologue is enhanced or produces.

The sequence of e. coli k12 b3498 (accession number NP_417955) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be oligopeptidase A.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary oligopeptidase A protein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be glycerol-3-phosphate and/or the glyceryl ester that comprises glycerol-3-phosphate, lipid, oils and/or fat, particularly increase glycerol-3-phosphate, the preferred quantity of the glycerol-3-phosphate of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b3498 is enhanced.In one embodiment, in the methods of the invention, oligopeptidase A activity of proteins is enhanced or produces, and for example the activity from colibacillary oligopeptidase A protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b3644 (accession number NP_418101) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the protein of the stress-induced that do not characterize.Therefore, in one embodiment, the inventive method comprises as shown here from the protein of the colibacillary stress-induced that does not characterize or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be glycerine and/or the triglyceride level that comprises glycerine, lipid, oils and/or fat, particularly increase glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, the activity of proteins of the stress-induced of Biao Zhenging is not enhanced or produces, and for example is enhanced or produces from the protein of the colibacillary stress-induced that does not characterize or the activity of its homologue.

The sequence of e. coli k12 b4073 (accession number NP_418497) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be formic acid dependent form nitrate reductase.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary formic acid dependent form nitrate reductase zymoprotein or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be glycerol-3-phosphate and/or the glyceryl ester that comprises glycerol-3-phosphate, lipid, oils and/or fat, particularly increase glycerol-3-phosphate, the preferred quantity of the glycerol-3-phosphate of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b4073 is enhanced.In one embodiment, in the methods of the invention, the activity of formic acid dependent form nitrate reductase is enhanced or produces, and for example the activity from colibacillary formic acid dependent form nitrate reductase or its homologue is enhanced or produces.

The sequence of e. coli k12 YHR072W (accession number NP_011939) has been published in Goffeau etc., Science 274 (5287), Science265 (5181) such as 546-547 (1996) and Johnston, 2077-2082 (1994), and it is 2 that its activity is defined as, 3-epoxy squalene-lanosterol cyclase protein matter.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary 2, the purposes of 3-epoxy squalene-lanosterol cyclase protein matter or its homologue, it is used for producing fine chemicals in biological or its part as described, be glycerine and/or the triglyceride level that comprises glycerine, lipid, oils and/or fat, particularly increase glycerine, the preferred quantity of the glycerine of free or combining form.In one embodiment, in the methods of the invention, 2, the activity of 3-epoxy squalene-lanosterol cyclase protein matter is enhanced or produces, for example from colibacillary 2, the activity of 3-epoxy squalene-lanosterol cyclase protein matter or its homologue is enhanced or produces.

The homologous compound (=homologue) of [0023.0.21.21] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, Table II the 3rd row, the 173rd row and 630-632 capable shown in any one homologue of polypeptide be to have same or similar active homologue.Particularly, each fine chemicals in the biology is given in active raising, preferably glycerine-3-phosphorus acid content increases.

In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 262-274,628 and 629 row.Particularly, each fine chemicals in the biology, preferably glycerine and/or total lipid content are given in active raising increases.

The homologue of polypeptide shown in Table II the 3rd row, the 173rd row and 630-632 are capable can be have increase glycerol-3-phosphate content and/or quantity is active, by Table I the 7th row, the 173rd row and 630-632 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, the 173rd row and 630-632 capable shown in polypeptide.

The homologue of polypeptide shown in Table II the 3rd row, 262-267,269,271-274 are capable can be have increase glycerine and/or total lipid content and/or quantity is active, by Table I the 7th row, 262-267,269,271-274 and 628-632 capable shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 262-267,269,271-274 and 628-632 capable shown in polypeptide.

[0023.1.0.21] as the homologue of the polypeptide shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable can be by Table I the 7th row, the 173rd, 262-274 and 628-632 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, the 173rd, 262-274 and 628-632 capable shown in polypeptide.

As the homologue of the polypeptide shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable can be by Table I the 7th row, the 173rd, 262-274 and 628-632 capable shown in nucleic acid molecule encoding polypeptide or can be Table II the 7th row, the 173rd, 262-274 and 628-632 capable shown in polypeptide.

[0024.0.0.21] sees [0024.0.0.0] for disclosing of this paragraph

[0025.0.21.21] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause in biology or its part, the preferred described biomass cells glycerol-3-phosphate and/or glycerine and/or total lipid level to increase, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II the 3rd row, the 173rd, 262-274 and 628-632 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, the 173rd, 262-274 and 628-632 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, the 173rd, any one proteinic biology or the enzyme activity shown in 262-274 and 628-632 are capable, if promptly with Table II the 3rd row, the 262nd and 263 and 628 and 630-632 capable shown in e. coli k12 protein or the 173rd, shown in 264-274 is capable with 629 in the yeast saccharomyces cerevisiae protein any one compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as each fine chemicals of increase in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

Above [0025.1.0.21] and [0025.2.0.21] sees for disclosing of [0025.1.0.21] and [0025.2.0.21] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.17] to [0033.0.0.17] saw for disclosing of [0026.0.0.17] to [0033.0.0.17] these paragraphs

[0034.0.21.21] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, its have Table II the 3rd row, the 173rd, 262-274 and protein shown in 628-632 is capable or by the active protein expression level of the protein of Table I the 5th row, the 173rd, 262-274 and nucleic acid molecule encoding shown in 628-632 is capable or its homologue (as Table I the 7th row, the 173rd, 262-274 and homologue shown in 628-632 is capable) or active aspect different, and its difference aspect biological chemistry or genetics reason.Therefore it shows each fine chemicals quantity that increases.

[0035.0.0.0] was to [0044.0.0.0] above [0035.0.0.21] to [0044.0.0.21] saw for disclosing of [0035.0.0.21] to [0044.0.0.21] these paragraphs

[0045.0.21.21] in one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YDR065W or its homologue (for example shown in Table II the 5th or 7 row, the 173rd row) is enhanced, preferably, give in one embodiment each fine chemicals, preferably glycerine-3-phosphoric acid be increased in 18% and 114% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2441 or its homologue (for example shown in Table II the 5th or 7 row, the 262nd row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred polarity part be increased in 41% and 81% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b3457 or its homologue (for example shown in Table II the 5th or 7 row, the 263rd row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred lipid part be increased in 18% and 44% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YBR084W or its homologue (for example shown in Table II the 5th or 7 row, the 264th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred polarity part be increased in 102% and 253% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YDR513W or its homologue (for example shown in Table II the 5th or 7 row, the 265th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred lipid part be increased in 17% and 48% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YDR513W or its homologue (for example shown in Table II the 5th or 7 row, the 265th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred polarity part be increased in 40% and 200% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YGL237C or its homologue (for example shown in Table II the 5th or 7 row, the 266th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred lipid part be increased in 23% and 73% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YIL150C or its homologue (for example shown in Table II the 5th or 7 row, the 267th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred lipid part be increased in 94% and 219% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR082C or its homologue (for example shown in Table II the 5th or 7 row, the 268th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred polarity part be increased in 41% and 173% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YRL224W or its homologue (for example shown in Table II the 5th or 7 row, the 269th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred lipid part be increased in 17% and 43% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR255C or its homologue (for example shown in Table II the 5th or 7 row, the 270th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred polarity part be increased in 44% and 73% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YMR015C or its homologue (for example shown in Table II the 5th or 7 row, the 271st row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred lipid part be increased in 18% and 23% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YOR344C or its homologue (for example shown in Table II the 5th or 7 row, the 272nd row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred lipid part be increased in 18% and 104% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YPL099C or its homologue (for example shown in Table II the 5th or 7 row, the 273rd row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred polarity part be increased in 55% and 104% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YPL268W or its homologue (for example shown in Table II the 5th or 7 row, the 274th row) is enhanced, preferably, give in one embodiment glycerine in each fine chemicals, the preferred polarity part be increased in 38% and 115% or more between.

At e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of the protein of the stress-induced that does not characterize (for example shown in Table II the 5th or 7 row, the 628th row) is enhanced, preferably give glycerine in the polarity part be increased in 17% and 44% or more between.

At yeast saccharomyces cerevisiae protein YHR072W or its homologue, for example 2, under the activity situation about being enhanced of 3-epoxy squalene-lanosterol cyclase protein matter (for example shown in Table II the 5th or 7 row, the 629th row), preferably give glycerine in the polarity part be increased in 19% and 109% or more between.

At e. coli k12 protein b2710 or its homologue, for example flavine rubredoxin (FIRd) bi-functional NO and O ₂Under the activity situation about being enhanced of reductase enzyme (for example shown in Table II the 5th or 7 row, the 630th row), preferably, give in one embodiment fine chemicals, preferably glycerine-3-phosphoric acid be increased in 20% and 95% or more between.

At e. coli k12 protein b3498 or its homologue, for example under the activity situation about being enhanced of oligopeptidase A (for example shown in Table II the 5th or 7 row, the 631st row), preferably, give in one embodiment fine chemicals, preferably glycerine-3-phosphoric acid be increased in 20% and 56% or more between.

At e. coli k12 protein b4073 or its homologue, for example under the activity situation about being enhanced of formic acid dependent form nitrate reductase (for example shown in Table II the 5th or 7 row, the 632nd row), preferably, give in one embodiment fine chemicals, preferably glycerine-3-phosphoric acid be increased in 49% and 145% or more between.

In addition, in vegetable cell or particularly plant seed, yeast saccharomyces cerevisiae protein YDR513W, YGL237C, YIL150C, YLR224W, YMR015C, YOR344C or YHR072W or e. coli k12 protein b3457, b3644, b2710, b3498 or b4073 or its homologue (for example Table II the 5th or 7 row, the 265th, 266,267,269,271,272 or 263 or 628-632 capable shown in) activity situation about being enhanced under, giving total lipid content increases.

[0046.0.0.21] in one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YDR065W or its homologue is enhanced, preferably giving the fine chemicals glycerol-3-phosphate increases.

In one embodiment, at e. coli k12 protein b2441 or its homologue, for example have under the situation that the activity of proteins of thanomin deaminase active is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at e. coli k12 protein b3457 or its homologue, for example have under the situation that the activity of proteins of branched-chain amino acid transport activity is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR084W or its homologue, for example have under the situation that the activity of proteins of C1-tetrahydrofolic acid (THFA) synthase activity is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR513W or its homologue, for example under the situation that the activity of glutathione reductase is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YGL237C or its homologue, for example under the situation that the transcriptional activation agent of vrg genes expression and the active activity of overall adjustment thing are enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YLR082C or its homologue, for example under the situation that the activity of the invalid lethal arrestin of Rad53 is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR224W or its homologue is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR255C or its homologue is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YMR015C or its homologue, for example have under the situation that the active activity of proteins of C-22 sterol desaturase is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR344C or its homologue, for example under the situation that the activity of proteins that is rich in Serine of involved in sugar glycolysis genetic expression is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YPL099C or its homologue, for example under the situation that the activity of Jia Ding membranin is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YPL268W or its homologue, for example have under the situation that the activity of proteins of phosphoinositide phospholipase activity is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at e. coli k12 protein b3644 or its homologue, for example under the situation that the activity of proteins of the stress-induced that does not characterize is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YHR072W or its homologue, for example 2, under the situation that the activity of 3-epoxy squalene-lanosterol cyclase protein matter is enhanced, preferably giving fine chemicals glycerine increases.

In one embodiment, at e. coli k12 protein b2710 or its homologue, for example flavine rubredoxin (FIRd) bi-functional NO and O ₂Under the situation that the activity of reductase enzyme is enhanced, preferably giving the fine chemicals glycerol-3-phosphate increases.

In one embodiment, at e. coli k12 protein b3498 or its homologue, for example under the situation that the activity of oligopeptidase A is enhanced, preferably giving the fine chemicals glycerol-3-phosphate increases.

In one embodiment, at e. coli k12 protein b4073 or its homologue, for example under the situation that the activity of formic acid dependent form nitrate reductase is enhanced, preferably giving the fine chemicals glycerol-3-phosphate increases.

Above [0047.0.0.21] and [0048.0.0.21] sees for disclosing of [0047.0.0.21] and [0048.0.0.21] these paragraphs [0047.0.0.0] and [0048.0.0.0]

[0049.0.21.21] has to give and improves each fine chemicals glycerol-3-phosphate quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise shown in SEQ ID NO:15671-15676 or 97963-97965 or 98284-98287 or 98529-98532 or Table IV the 7th row, the 173rd or 630-632 capable shown in consensus sequence sequence or as Table II the 5th or 7 row, the 173rd or 630-632 capable shown in its function homologue described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 173rd or 630-632 capable shown in nucleic acid molecule or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the glycerol-3-phosphate level of giving described in the literary composition increases.

Have to give and improve each fine chemicals glycerine and/or total fat quantity or the active protein of level and preferably have the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as SEQ ID NO:28565-28575 or 28576,28577 or 28578 or 28579-28581 or 28582-28584 or 28585,28586 or 28587-28590 or 28591-28594 or 28595-28597 or 28598-28600 or 28601-28605 or 98450-98452 or 98703-98713 shown in or Table IV the 7th row, the sequence of consensus sequence shown in 262-274 or 628 and 629 row or as Table II the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in 262-274 or 628 and 629 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in 262-274 or 628 and 629 row) sequence of coded polypeptide, and have the activity that the glycerine level of giving described in the literary composition increases.

[0050.0.21.21] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, the sylvite of glycerol-3-phosphate or sodium salt, perhaps their ester such as its single acyl lipid acid or two acyl lipid acid.

[0051.0.21.21] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise the composition of glycerine and/or glycerol-3-phosphate and/or total fat.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of glycerine and/or glycerol-3-phosphate and/or total fat.

[0052.0.0.21] sees [0052.0.0.0] for disclosing of this paragraph

[0053.0.21.21] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention (for example having as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in)) and is expressed and increase, and has the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention (for example having as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in)) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in), perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in);

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having) as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide (for example having as the active polypeptide of protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in)).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.21.21] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example give each fine chemicals after the active expression of polypeptides of protein or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in polypeptide) or the activity shown in capable and increase improving coded protein or have as Table II the 3rd or 5 row, the 173rd, 262-274 and 628-632.

[0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.21] to [0067.0.0.21] saw for disclosing of [0055.0.0.21] to [0067.0.0.21] these paragraphs

[0068.0.21.21] is can being not that the generation of glycerine and/or glycerol-3-phosphate and/or total fat causes the mode of disadvantageous effect to introduce sudden change to fine chemicals.

[0069.0.0.21] sees [0069.0.0.0] for disclosing of this paragraph

[0070.0.21.21] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, the composition of for example favourable glycerol-3-phosphate and glycerine or their biological chemistry derivative for example contains the composition of high level (from the physiology of nutrition angle) glycerol-3-phosphate and glycerine or their derivative (comprising total fat).

[0071.0.0.21] sees [0071.0.0.0] for disclosing of this paragraph

[0072.0.0.21]％

[0073.0.21.21] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of polypeptide of the present invention or its homologue (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation.

[0074.0.21.21] sees [0071.0.0.17] for disclosing of this paragraph

[0075.0.0.0] was to [0077.0.0.0] above [0075.0.0.21] to [0077.0.0.21] saw for disclosing of [0075.0.0.21] to [0077.0.0.21] these paragraphs

[0078.0.21.21] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes.Method well known to those skilled in the art be can use and purified fermentation broth, tunning, plant or plant product come.For these different methods of setting up gradually, product is glycerine and/or glycerol-3-phosphate and/or as the glycerine of lipid component or comprise glycerine and/or glycerol-3-phosphate and/or as the composition of the glycerine of lipid component, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.0] was to [0084.0.0.0] above [0079.0.0.21] to [0084.0.0.21] saw for disclosing of [0079.0.0.21] to [0084.0.0.21] these paragraphs

[0085.0.21.21] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as Table I the 5th or 7 row, the 173rd, 262-274 and nucleotide sequence or derivatives thereof shown in 628-632 is capable, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

Above [0086.0.0.21] and [0087.0.0.21] sees for disclosing of [0086.0.0.21] and [0087.0.0.21] these paragraphs [0086.0.0.0] and [0087.0.0.0]

[0088.0.21.21] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Stronger and output is enhanced to the resistance of biological and abiotic stress because the nutritive value that for example is used for the plant of raise poultry depends on above-mentioned fine chemicals or plant, so this is very important for the plant breeder.

[0088.1.0.21] sees [0088.1.0.0] for disclosing of this paragraph

[0088.0.0.0] was to [0094.0.0.0] above [0089.0.0.21] to [0094.0.0.21] saw for disclosing of [0089.0.0.21] to [0094.0.0.21] these paragraphs

[0095.0.21.21] advantageously increases glycerine and/or glycerol-3-phosphate and/or total fat pond to separate a large amount of pure basically fine chemicals and/or to obtain the resistance of the raising of biological and abiotic stress and obtain high yield by method of the present invention in genetically modified organism.

[0096.0.21.21] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.

[0097.0.0.21] sees [0097.0.0.0] for disclosing of this paragraph

[0098.0.21.21] in preferred embodiments, each fine chemicals be produce according to the present invention and carry out isolating where necessary.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.21.21].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition that comprises each fine chemicals.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.21.21] comprises the transgenic plant that the method according to this invention synthetic comprises fine chemicals such as glycerine and/or glycerol-3-phosphate and/or total fat and can advantageously directly put on market, and do not need institute's synthetic fine chemicals is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet each fine chemicals that the method according to this invention produces can also be as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free fine chemicals from biology, advantageously separate from plant.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Because glycerine and/or glycerol-3-phosphate and/or total fat are positioned at cell in the microorganism, therefore must relate to separation to biomass to their recovery.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.

[0101.0.21.21] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.21.21] for example, glycerine and/or glycerol-3-phosphate can separate and MS (mass spectrum) detection method is advantageously analyzed by HPLC, LC or GC.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect the existence of the product that contains glycerine and/or glycerol-3-phosphate to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

Can pass through as Bligh and Dyer (Can J Biochem Phys 1959,37,911-917) described lipid extraction method is advantageously analyzed the total fat in the plant seed, pass through then as Benning and Sommerville (J Bacteriol 1992,174, the 6479-6487) content of the gas Chromatographic Determination lipid described in.

[0103.0.21.21] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as the sequence shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as the sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer as sequence shown in Table III the 7th row, the 173rd, 262-274 and 628-632 are capable obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, the 173rd, 262-267,269 and 271-274 and 628-632 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.21.21] in one embodiment, the sequence difference was one or more Nucleotide shown in nucleic acid molecule of the present invention and Table I A the 5th or 7 row, the 173rd, 262-274 and 628-632 were capable.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I A the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II A the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in polypeptide of sequence.

[00103.2.21.21] in one embodiment, the sequence difference was one or more Nucleotide shown in nucleic acid molecule of the present invention and Table I B the 5th or 7 row, the 173rd, 262-274 and 628-632 were capable.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I B the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II B the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in polypeptide of sequence.

[0104.0.21.21] in one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I A the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sequence form.In one embodiment, the identity of sequence was less than 100%, 99.999%, 99.99%, 99.9% or 99% shown in nucleic acid molecule of the present invention and Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 were capable, preferably, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table II A the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in polypeptide of sequence.

[0105.0.0.0] was to [0107.0.0.0] above [0105.0.0.21] to [0107.0.0.21] saw for disclosing of [0105.0.0.21] to [0107.0.0.21] these paragraphs

Advantageously improved in [0108.0.21.21] method of the present invention and had Table I the 5th or 7 row, the 173rd, the nucleic acid molecule of sequence shown in 262-274 and 628-632 are capable, from Table II the 5th or 7 row, the 173rd, aminoacid sequence shown in 262-274 and 628-632 are capable is derived or is listed as from containing Table IV the 7th, the 173rd, 262-267,269 and 271-274 and 628-632 capable shown in the polypeptide deutero-nucleic acid molecule of consensus sequence, perhaps its coding has as Table II the 3rd, 5 or 7 row, the 173rd, enzymic activity of polypeptide shown in 262-274 and 628-632 are capable or bioactive polypeptide or for example to give each fine chemicals behind its expression or active the increasing be derivative or the homologue that glycerine and/or glycerol-3-phosphate and/or total fat increase.

[0109.0.21.21] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make that each fine chemicals, particularly glycerine that the inventive method produced and/or glycerol-3-phosphate and/or total fat are optimized and synthesize.

[0110.0.0.21] help the inventive method and coding have the inventive method or the used polypeptide of process of the present invention (for example Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in protein or by Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in coded protein or its homologue of nucleic acid molecule, shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable) nucleic acid molecule of active polypeptide can determine from generally open database.

[0111.0.0.21] sees [0111.0.0.0] for disclosing of this paragraph

The nucleic acid molecule that uses in [0112.0.21.21] the inventive method is the isolated nucleic acid sequences form, its coding has as the polypeptide of polypeptide active shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable or has polypeptide as peptide sequence shown in Table II the 5th and 7 row, the 173rd, 262-274 and 628-632 are capable, and gives glycerine and/or glycerol-3-phosphate and/or total lipid level and increase.

Above [0113.0.0.21] to [0120.0.0.21] sees for disclosing of [0113.0.0.21] to [0120.0.0.21] these paragraphs [0113.0.0.0] and [0120.0.0.0]

[0121.0.21.21] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, peptide sequence or its functional homologue as herein described had the difference of one or more amino acid moleculars shown in the 173rd row and 630-632 were capable, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, giving the glycerol-3-phosphate level after the activity of peptide sequence shown in the 173rd row and 630-632 are capable increases, and is perhaps improving Table II the 5th or 7 row, give glycerine after the activity of peptide sequence shown in capable and the 628th and 629 row of 262-274 and/or total lipid level increases.

Above [0122.0.0.21] to [0127.0.0.21] sees for disclosing of [0122.0.0.21] to [0127.0.0.21] these paragraphs [0122.0.0.0] and [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.21.21] polymerase chain reaction (PCR) amplification is used (for example as the primer shown in Table III the 7th row, the 173rd, 262-274 and 628-632 are capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sequence or from producing as sequence deutero-sequence shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.

[0129.0.21.21] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, the 173rd, 262-267,269 and 271-274 and 628-632 capable shown in consensus sequence compare since then.

[0130.0.21.21] can use then degenerated primer by pcr amplification have aforementioned activity (as improve to express or activity after give glycerine and/or glycerol-3-phosphate and/or total fat and increase) the new protein fragment or comprise as described in segmental protein.

[0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.21] to [0138.0.0.21] saw for disclosing of [0131.0.0.21] to [0138.0.0.21] these paragraphs

[0139.0.21.21] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with for the Table I the 5th of glycerol-3-phosphate or 7 row, the 173rd row and 630-632 is capable or be listed as for the Table I the 5th of glycerine or 7, capable and the 628th and 629 row of 262-274, preferably the Table I B the 5th or 7 for total fat is listed as, the 173rd row and 630-632 is capable or 262-274 is capable and the 628th and 629 capable shown in sequence hybridization, and the coding expression has each fine chemicals of increasing, i.e. glycerine and/or glycerol-3-phosphate and/or the always active peptide of fat.

[0140.0.0.0] was to [0146.0.0.0] above [0140.0.0.21] to [0146.0.0.21] saw for disclosing of [0140.0.0.21] to [0146.0.0.21] these paragraphs

[0147.0.21.21] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I B the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.21.21] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, preferred Table I B the 5th or 7 row, the 173rd, nucleotide sequence shown in 262-274 and 628-632 are capable or its portion homologous are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases glycerine and/or glycerol-3-phosphate and/or total fat.

[0149.0.21.21] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, preferred Table I B the 5th or 7 row, the 173rd, one of nucleotide sequence shown in 262-274 and 628-632 are capable or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding have above-mentioned activity (as give glycerol-3-phosphate and/or glycerine increase) and randomly as Table II the 5th be listed as, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 5th or 7 row, the 173rd, the protein of protein active shown in 262-274 and 628-632 are capable.

[00149.1.21.21] randomly, in one embodiment, with Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I B the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity is for as Table II the 3rd row, the 173rd, 262-274 with 628-632 is capable, preferred Table II B the 5th or 7 row, the 173rd, 262-274 and the 628-632 protein shown in capable is known activity or is used for these protein of note.

[0150.0.21.21] in addition, nucleic acid molecule of the present invention can only contain Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I B the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give glycerol-3-phosphate and/or glycerine increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sense strand, Table I the 5th or 7 row, the 173rd, 262-274 and the 628-632 of one of sequence capable shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, the 173rd, 262-274 and 628-632 capable shown in the right PCR of primer will produce as Table I the 5th or 7 be listed as, the 173rd, 262-274 and 628-632 capable shown in fragment or its gene product of polynucleotide sequence.Preferably Table I B the 7th row, the 173rd, 262-274 and 628-632 are capable.

[0151.0.0.21]: see [0151.0.0.0] for disclosing of this paragraph

[0152.0.21.21] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and are listed as Table II the 5th or 7, the 173rd, the abundant homology of aminoacid sequence shown in 262-274 and 628-632 are capable, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or raising glycerol-3-phosphate as be shown in the examples (the 173rd row and 630-632 are capable) or glycerine (capable and the 628th and 629 row of 262-274) and total fat the (the 265th, 266,267,269,271,272 and 263 row) activity of level.

[0153.0.21.21] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as the amino-acid residue identical or of equal value of aminoacid sequence minimal number shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as protein shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its part have Table II the 3rd row for example as herein described, the 173rd, 262-274 and 628-632 capable shown in the activity of polypeptide.

[0154.0.21.21] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein be at least about 30%, 35%, 45% or 50% as complete amino acid sequence homology shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

Above [0155.0.0.21] and [0156.0.0.21] sees for disclosing of [0155.0.0.21] and [0156.0.0.21] these paragraphs [0155.0.0.0] and [0156.0.0.0]

[0157.0.21.21] the present invention relate in addition owing to the genetic code degeneracy be different from Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in one of nucleotide sequence (with its part) and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase) for example comprises as polypeptide of sequence shown in Table IV the 7th row, the 173rd, 262-274 and 628-632 are capable or as the nucleic acid molecule of polypeptide shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its function homologue.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as Table IV the 7th row, the 173rd, 262-267,269 and the consensus sequence of 271-274 shown in capable or as the aminoacid sequence of polypeptide shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 7th row, the 173rd, 262-267,269 and the consensus sequence of 271-274 shown in capable or as the basic homology of aminoacid sequence of polypeptide shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I A the 5th or 7 row, the 173rd, 262-274 and the 628-632 sequence shown in capable.Preferably, nucleic acid molecule of the present invention is the functional homologous compound of Table I B the 7th row, the 173rd, 262-274 and nucleic acid molecule shown in 628-632 is capable or identical with it.

[0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.21] to [0160.0.0.21] saw for disclosing of [0158.0.0.21] to [0160.0.0.21] these paragraphs

[0161.0.21.21] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.21] sees [0162.0.0.0] for disclosing of this paragraph

[0163.0.21.21] preferably, under stringent condition with Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals quantity increase in biology or its part (for example tissue, cell or cell chamber) after using activity of proteins).

[0164.0.0.21] sees [0164.0.0.0] for disclosing of this paragraph

[0165.0.21.21] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sequence).

Above [0166.0.0.21] and [0167.0.0.21] sees for disclosing of [0166.0.0.21] and [0167.0.0.21] these paragraphs [0166.0.0.0] and [0167.0.0.0]

[0168.0.21.21] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are with different as the contained sequence of sequence shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, but have kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the aminoacid sequence shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, the 173rd, 262-274 is identical at least about 60% with the sequence shown in 628-632 is capable, more preferably with as Table II the 5th or 7 be listed as, the 173rd, 262-274 is identical at least about 70% with one of sequence shown in 628-632 is capable, even more preferably with as Table II the 5th or 7 be listed as, the 173rd, sequence shown in 262-274 and 628-632 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, the 173rd, sequence shown in 262-274 and 628-632 are capable is at least about 96%, 97%, 98% or 99% is identical.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table II B the 7th row, the 173rd, 262-274 and 628-632 capable shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, the 173rd, 262-274 with 628-632 is capable, preferred Table II B the 7th row, the 173rd, 262-274 and the 628-632 aminoacid sequence shown in capable is at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 7th row, the 173rd, 262-274 is identical at least about 60% with the sequence shown in 628-632 is capable, more preferably with as Table II the 5th or 7 be listed as, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 7th row, the 173rd, 262-274 is identical at least about 70% with one of sequence shown in 628-632 is capable, even more preferably with as Table II the 5th or 7 be listed as, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 7th row, the 173rd, sequence shown in 262-274 and 628-632 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 7th row, the 173rd, sequence shown in 262-274 and 628-632 are capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.0] was to [0172.0.0.0] above [0169.0.0.21] to [0172.0.0.21] saw for disclosing of [0169.0.0.21] to [0172.0.0.21] these paragraphs

[0173.0.21.21] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:27329 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:27329 sequence before use.

[0174.0.0.21]: see [0174.0.0.0] for disclosing of this paragraph

[0175.0.21.21] for example, the sequence that has 80% homology at protein level and SEQ ID NO:27330 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:27330 sequence.

[0176.0.21.21] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the 173rd, the functional equivalent that one of polypeptide obtained shown in 262-274 and 628-632 were capable be listed as according to of the present invention as Table II the 5th or 7, the 173rd, one of polypeptide had at least 30% shown in 262-274 and 628-632 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, the 173rd, polypeptide shown in 262-274 and 628-632 are capable has essentially identical character and discerns.

[0177.0.21.21] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, the 173rd, 262-274 and 628-632 are capable, preferred Table I B the 173rd, the functional equivalent that nucleotide sequence obtained shown in 262-274 and 628-632 were capable be listed as according to of the present invention as Table II the 5th or 7, the 173rd, one of polypeptide had at least 30% shown in 262-274 and 628-632 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 173rd, the polypeptide of the essentially identical character of polypeptide shown in 262-274 and 628-632 are capable.

[0178.0.0.21] sees [0178.0.0.0] for disclosing of this paragraph

[0179.0.21.21] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, the 173rd, 262-274 and 628-632 are capable, preferred Table I B the 173rd, shown in 262-274 and 628-632 are capable) the middle replacement of introducing one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 173rd, the nucleic acid molecule of the homologue of protein sequence shown in 262-274 and the 628-632.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, the 173rd, 262-274 with 628-632 is capable, preferred Table I B the 173rd, 262-274 and 628-632 introduce sudden change in the encoding sequence shown in capable.

[0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.21] to [0183.0.0.21] saw for disclosing of [0180.0.0.21] to [0183.0.0.21] these paragraphs

[0184.0.21.21] is employed to have as Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, preferred Table I B the 173rd, the nucleotide sequence homologous compound of sequence shown in 262-274 and 628-632 are capable, perhaps come Table II the 5th or 7 row freely, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 173rd, the homologous compound of the nucleotide sequence of sequence shown in 262-274 and 628-632 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I B the 173rd, 262-274 and 628-632 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.21.21] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as Table I the 5th or 7 row, the 173rd, 262-274 with 628-632 is capable, preferred Table I B the 173rd, 262-274 and the 628-632 sequence shown in capable.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, the 173rd, 262-274 with 628-632 is capable, other Nucleotide of not showing in preferred Table I B the 173rd, 262-274 and the 628-632 arbitrary sequence shown in capable.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, the 173rd, 262-274 with 628-632 is capable, preferred Table I B the 173rd, 262-274 and the 628-632 sequence shown in capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.21.21] also preferred the inventive method comprise as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table II B the 173rd, 262-274 and 628-632 capable shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table II B the 173rd, 262-274 and 628-632 capable shown in sequence identical.

[0187.0.21.21] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table II B the 173rd, 262-274 and 628-632 capable shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table II B the 173rd, 262-274 and 628-632 capable shown in the encoding sequence of sequence identical.

[0188.0.21.21] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active comparing with polypeptide expressed activity as Table II the 5th or 7 row, the 173rd, 262-274 and shown in 628-632 is capable and under the same conditions do not reduced substantially.

In one embodiment, polypeptide of the present invention be comprise Table II B the 7th row, the 173rd, 262-274 and 628-632 capable shown in sequence or by its homologue of forming.

[0189.0.21.21] as the homologous compound of sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.21]: see [0190.0.0.0] for disclosing of this paragraph

[0191.0.21.21] compares with biological or its part of described contrast or selection in one embodiment, according to the inventive method described herein should biology or its part each fine chemicals level of producing protein bound improve.

[0191.1.0.21]: see [0191.1.0.0] for disclosing of this paragraph

[0192.0.0.0] was to [0203.0.0.0] above [0192.0.0.21] to [0203.0.0.21] saw for disclosing of [0192.0.0.21] to [0203.0.0.21] these paragraphs

[0204.0.21.21] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table II B the 173rd, 262-274 and 628-632 polypeptide or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly glycerine (the 265th, 266,267,269,271, the 272 and 263 row) quantity of glycerol-3-phosphate (the 173rd row and 630-632 are capable) or glycerine (capable and the 628th and 629 row of 262-274) and the total fat composition of conduct increases;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I B the 173rd, 262-274 and 628-632 nucleic acid molecule or its segmental nucleic acid molecule shown in capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises the nucleic acid molecule that as the primer shown in Table III the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or primer amplification from cDNA library or genomic library is obtained by using, and in biological or its part, give each fine chemicals, i.e. the increase of glycerine (the 265th, 266,267,269,271, the 272 and 263 row) quantity of glycerol-3-phosphate (the 173rd row and 630-632 are capable) or glycerine (capable and the 628th and 629 row of 262-274) and total fat composition;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, the 173rd, 262-267,269 and 271-274 is capable and 628-632 capable shown in consensus sequence and in biological or its part, give each fine chemicals, i.e. glycerol-3-phosphate (the 173rd row and 630-632 are capable) or glycerine (capable and the 628th and 629 row of 262-274) and as the nucleic acid molecule of glycerine (the 265th, 266,267,269,271,272 and 263 row) the quantity increase of total fat composition;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be glycerol-3-phosphate (the 173rd row capable of 630-632) or glycerine (capable and the 628th and 629 row of 262-274) and the nucleic acid molecule that increases as glycerine (the 265th, 266,267,269,271, the 272 and 263 row) quantity of total fat composition, described peptide coding is as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, the preferred structural domain of Table II B the 173rd, 262-274 and the 628-632 polypeptide shown in capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k=) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, the 173rd, 262-274 and 628-632 are capable, preferred Table I B the 173rd, nucleic acid molecule shown in 262-274 and 628-632 are capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, preferred Table II B the 173rd, at least the 15nt of the nucleic acid molecule of polypeptide shown in 262-274 and 628-632 are capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A or IB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention do not form by Table I A or IB the 5th or 7 row, the 173rd, 262-274 and 628-632 by the sequence shown in capable.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as the peptide sequence shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as the protein of sequence shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable at least 30% identical and with Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

[0205.0.0.21] to [0206.0.0.21]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.21.21] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of glutamic acid metabolism, phosphoenolpyruvic acid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.0] was to [0226.0.0.0] above [0208.0.0.21] to [0226.0.0.21] saw for disclosing of [0208.0.0.21] to [0226.0.0.21] these paragraphs

[0227.0.21.21] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, the 173rd, 262-274 and the capable sequence or derivatives thereof of mentioning of 628-632, can advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, the extra polypeptide of expressing other genes of at least one acetyl-CoA or malonyl--CoA pathways metabolism or having very-long-chain fatty acid acid acyl (VLCFA) CoA synthase activity in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.21.21] therefore cultivated in another embodiment of the present invention and crossed expression coding and glycerol-3-phosphate and/or glycerine metabolism (particularly glycerol-3-phosphate or glycerine are synthetic) at least one nucleic acid of related protein or the biology of a gene simultaneously.

[0229.0.21.21] can be coding lipid acid approach (other gene of acetyl-CoA or malonyl--CoA) or have other gene of the polypeptide of very-long-chain fatty acid acyl (VLCFA) CoA synthase activity for example with employed other the favourable nucleotide sequences of expressing as sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable and/or the combination of aforementioned biosynthesis gene of present method.These genes cause the synthetic increase of VLCFA.

[0230.0.0.21] sees [0230.0.0.0] for disclosing of this paragraph

[0231.0.21.21] is in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded glycerine and/or glycerol-3-phosphate and/or lipid simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.For example it be known to those skilled in the art that, to the inhibition of the enzyme of degraded glycerine and/or glycerol-3-phosphate or lipid or check and to cause in the plant glycerine and/or glycerol-3-phosphate and/or total fat accumulation to increase.

[0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.21] to [0276.0.0.21] saw for disclosing of [0232.0.0.21] to [0276.0.0.21] these paragraphs

[0277.0.21.21] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.

[0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.21] to [0282.0.0.21] saw for disclosing of [0278.0.0.21] to [0282.0.0.21] these paragraphs

[0283.0.21.21] in addition, can from cell, separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as protein shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable, perhaps as the antibody of polypeptide shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its antigen part, it can utilize polypeptide of the present invention or its fragment to produce by standard technique.Preferably specificity in conjunction with Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in polypeptide, more preferably specificity in conjunction with Table II the 5th row, the 173rd, 262-274 and 628-632 capable shown in the monoclonal antibody of polypeptide.

[0284.0.0.21] sees [0284.0.0.0] for disclosing of this paragraph

[0285.0.21.21] the present invention relates to have as sequence shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable in one embodiment.

[0286.0.21.21] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, the 173rd, 262-267,269 and 271-274 is capable and 628-632 capable shown in consensus sequence or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, the 173rd, 262-267,269 and 271-274 is capable and 628-632 capable shown in consensus sequence or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, the 173rd, 262-267,269 and 271-274 is capable and 628-632 capable shown in the polypeptide of consensus sequence (each row).

[0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.21] to [0290.0.0.21] saw for disclosing of [0287.0.0.21] to [0290.0.0.21] these paragraphs

[0291.0.21.21] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, the 173rd, sequence shown in 262-274 and 628-632 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, the 173rd, sequence shown in 262-274 and 628-632 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.

[0292.0.0.21] sees [0292.0.0.0] for disclosing of this paragraph

[0293.0.21.21] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.In another embodiment, polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide not by Table I A or IB the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.21.21] in one embodiment, the present invention relates to have as Table II the 3rd row, the 173rd, the polypeptide of activity of proteins shown in 262-274 and 628-632 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, the 173rd, sequence shown in 262-274 and 628-632 are capable.

[0295.0.0.0] was to [0297.0.0.0] above [0295.0.0.21] to [0297.0.0.21] saw for disclosing of [0295.0.0.21] to [0297.0.0.21] these paragraphs

The chemical of [00297.1.21.21] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, the 173rd, 262-274 and 628-632 capable shown in the active polypeptide of polypeptide.

[0298.0.21.21] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II the 5th or 7 row, the 173rd, 262-274 and 628-632.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.

[0299.0.21.21] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization (preferably hybridizing under above-mentioned stringent condition) that this nucleotide sequence and nucleic acid branch of the present invention give.Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% as one of nucleotide sequence sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable homology, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.

[0300.0.21.21] is therefore, and be described in detail as this paper, polypeptide of the present invention because natural variation or mutagenesis can be listed as on aminoacid sequence with as Table II the 5th or 7, the 173rd, 262-274 and 628-632 capable shown in sequence different.Therefore, this polypeptide contains with complete amino acid sequence homology as sequence shown in Table II A or IIB the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable and is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.21] sees [0301.0.0.0] for disclosing of this paragraph

The biologically-active moiety of [0302.0.21.21] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the 173rd, the aminoacid sequence of the aminoacid sequence shown in 262-274 and 628-632 are capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.21] sees [0303.0.0.0] for disclosing of this paragraph

[0304.0.21.21] operation nucleic acid molecule of the present invention may cause generation have basically as the active of polypeptide shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.0] was to [0308.0.0.0] above [0305.0.0.21] to [0308.0.0.21] saw for disclosing of [0305.0.0.21] to [0308.0.0.21] these paragraphs

[0306.1.0.21]％

[0309.0.21.21] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, the 173rd, protein shown in 262-274 and 628-632 are capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, the 173rd, " other polypeptide " during 262-274 and 628-632 are capable is meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, the 173rd, polypeptide shown in 262-274 and 628-632 are capable is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, the 173rd, it is proteinic and shown in 262-274 and 628-632 are capable from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, the 173rd, 262-274 and capable " other polypeptide " of 628-632 and do not give each fine chemicals increase in biology or its part.

[0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.21] to [0334.0.0.21] saw for disclosing of [0310.0.0.21] to [0334.0.0.21] these paragraphs

[0335.0.21.21] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.21] to [0342.0.0.21] saw for disclosing of [0336.0.0.21] to [0342.0.0.21] these paragraphs

[0343.0.21.21] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its homologue in a kind of organism.

[0344.0.0.0] was to [0361.0.0.0] above [0344.0.0.21] to [0361.0.0.21] saw for disclosing of [0344.0.0.21] to [0361.0.0.21] these paragraphs

[0362.0.21.21] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, the 173rd, polypeptide shown in 262-274 and 628-632 are capable, for example coding has protein such as Table II the 3rd row, the 173rd, the polypeptide of polypeptide active shown in 262-274 and 628-632 are capable) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.The transgenosis that has as the polypeptide of polypeptide active shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable is meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, the activity that is noted as polypeptide shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable (for example have as Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable shown in polypeptide of sequence) is enhanced.Example and the inventive method are described in above.

[0363.0.0.21] sees [0363.0.0.0] for disclosing of this paragraph

[0364.0.21.21] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---for example naturally occurring coding as the promotor of the gene of polypeptide of the present invention shown in Table II the 3rd row, the 173rd, 262-274 and 628-632 are capable with accordingly as the combination of protein coding sequence shown in Table I the 5th row, the 173rd, 262-274 and 628-632 are capable---becomes transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.21] to [0373.0.0.21] for [0365.0.0.21], disclosing of these paragraphs of-[0373.0.0.21] sees that top [0365.0.0.0] is to [0373.0.0.0]

[0374.0.21.21] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, glycerine that produces in the inventive method and/or glycerol-3-phosphate, particularly each fine chemicals also can be from plant with free glycerol and/or each fine chemicals of glycerol-3-phosphate, particularly free or be bonded to compound or the isolated in form of part (such as but not limited to monoacylglycerol, diacylglycerol or triacylglycerol, phosphoglyceride, monoacylglycerol phosphoric acid or diacylglycerol phosphoric acid).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

Above [0375.0.0.21] and [0376.0.0.21] sees for disclosing of [0375.0.0.21] and [0376.0.0.21] these paragraphs [0375.0.0.0] and [0376.0.0.0]

[0377.0.21.21] therefore the invention still further relates to the method according to this invention, separated the glycerine and/or glycerol-3-phosphate and/or the total fat that are produced thus.

[0378.0.21.21] can separate by this way and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than 90% the glycerine that produces in the method and/or glycerol-3-phosphate and/or total fat.As required, resultant glycerine and/or glycerol-3-phosphate and/or total fat can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.21.21] is the mixture of each fine chemicals glycerine, glycerol-3-phosphate and total fat by the product that the present invention produced in one embodiment.

Glycerine and/or glycerol-3-phosphate or the suitable synthetic parent material of total fat that [0380.0.21.21] obtains by carrying out the inventive method as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises glycerine that separation produces and/or glycerol-3-phosphate composition or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention be the glycerine that produces of the inventive method and/or glycerol-3-phosphate and/or total fat or genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine purposes or be used to produce the purposes of glycerine and/or glycerol-3-phosphate, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

Above [0381.0.0.21] and [0382.0.0.21] sees for disclosing of [0381.0.0.21] and [0382.0.0.21] these paragraphs [0381.0.0.0] and [0382.0.0.0]

[0383.0.21.21]％

[0384.0.0.21] sees [0384.0.0.0] for disclosing of this paragraph

The fermented liquid that [0385.0.21.21] obtains with this kind approach, particularly contain fermented liquid with other organic acid, amino acid, polypeptide or polysaccharide blended glycerine and/or glycerol-3-phosphate and/or total fat, usually dry matter content accounts for the 1-70% of weight, preferred 7.5 to 25%.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means and to utilize the concentration of sugar during this period of time to remain on or be reduced to 0-10g/l in the fermention medium, preferred to 0-3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.21.21] therefore can also be further purified the glycerine and/or the glycerol-3-phosphate that produce according to the present invention.For this reason, the composition that will contain product separates (by for example open column chromatography or HPLC), and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.21] to [0392.0.0.21] saw for disclosing of [0387.0.0.21] to [0392.0.0.21] these paragraphs

[0393.0.21.21] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 is capable, preferred Table I B the 5th or 7 row, the 173rd, 262-274 and the 628-632 nucleic acid molecule shown in capable is hybridized under lax stringent condition nucleic acid molecule, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.0] was to [0398.0.0.0] above [0394.0.0.21] to [0398.0.0.21] saw for disclosing of [0394.0.0.21] to [0398.0.0.21] these paragraphs

[0399.0.21.21] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving each fine chemicals generation raising under these conditions.

Whether [00399.1.21.21] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect and depends on as the active of the polypeptide shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its homologue or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high biology phenotype more much at one as activity of proteins shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable.

[0400.0.0.0] was to [0416.0.0.0] above [0400.0.0.21] to [0416.0.0.21] saw for disclosing of [0400.0.0.21] to [0416.0.0.21] these paragraphs

[0417.0.21.21] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor to glycerine and/or glycerol-3-phosphate biosynthetic pathway has resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block glycerine and/or glycerol-3-phosphate synthetic inhibitor.

[0418.0.0.0] was to [0423.0.0.0] above [0418.0.0.21] to [0423.0.0.21] saw for disclosing of [0418.0.0.21] to [0423.0.0.21] these paragraphs

[0424.0.21.21] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other organic acids.Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.0] was to [0434.0.0.0] above [0425.0.0.21] to [0434.0.0.21] saw for disclosing of [0425.0.0.21] to [0434.0.0.21] these paragraphs

[0435.0.21.21] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.21.21] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce glycerine and/or glycerol-3-phosphate.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that [0436.1.21.21] external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.21.21] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.21.21] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.21.21] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.21.21] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad 50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.0] was to [0443.0.0.0] above [0441.0.0.21] to [0443.0.0.21] saw for disclosing of [0441.0.0.21] to [0443.0.0.21] these paragraphs

[0444.0.21.21] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.21.21] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.0] was to [0450.0.0.0] above [0446.0.0.21] to [0450.0.0.21] saw for disclosing of [0446.0.0.21] to [0450.0.0.21] these paragraphs

Above [0451.0.5.21] sees for disclosing of this paragraph [0451.0.5.5]

Above [0452.0.0.21] and [0453.0.0.21] sees for disclosing of [0452.0.0.21] and [0453.0.0.21] these paragraphs [0452.0.0.0] and [0453.0.0.0]

[0454.0.21.21] embodiment 8: analyzing nucleic acid molecules is to the influence of glycerine and/or glycerol-3-phosphate generation

[0455.0.21.21] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example glycerine and/or glycerol-3-phosphate) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being glycerine and/or glycerol-3-phosphate) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia ofIndustrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry andMolecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recoveryand purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", JohnWiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recoveryprocesses for biological Materials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; The 1l chapter, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques inbiotechnology ", Noyes Publications).

[0456.0.0.21]: see [0456.0.0.0] for disclosing of this paragraph

[0457.0.21.21] embodiment 9: the purifying of glycerine and/or glycerol-3-phosphate

[0458.0.21.21] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS, GC-MS or TLC can clearly detect the existence of glycerine and/or glycerol-3-phosphate to the biology analysis of recombinating.

It is biological to use the following step to analyze, as the total amount that produces in the inventive method used yeast: by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000 W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 4022S1V diluter with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.20ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, Hewlett Packard 1100 HPLC have been used, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Tech nologies, can be from Ultra Scientific Inc., 250 Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6 * 250mm[Waters, 34 Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

[0459.0.21.21] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified glycerine and/or glycerol-3-phosphate.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.0.21] sees [0460.0.0.0] for disclosing of this paragraph

[0461.0.21.21] embodiment 10: clone SEQ ID NO:14364,27329,27377,27857,28009,28145,28179,28197,28201,28207,28211,28341,28363,28369,98288,98533,97771,97966 or 98453 is used for expressing plant

[0462.0.0.21] sees [0462.0.0.0] for disclosing of this paragraph

[0463.0.21.21] passes through pcr amplification SEQ ID NO:14364,27329,27377,27857,28009,28145,28179,28197,28201,28207,28211,28341,28363,28369,98288,98533,97771,97966 or 98453 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.0] was to [0466.0.0.0] above [0464.0.0.21] to [0466.0.0.21] saw for disclosing of [0464.0.0.21] to [0466.0.0.21] these paragraphs

[0466.1.0.21] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.21.21] selects following primer sequence for genes of SEQ ID NO:14364:

I) forward primer (SEQ ID NO:14370)

atggctcaaa?attttggaaa?gattcc

Ii) reverse primer (SEQ ID NO:14371)

ttaaaaaccg?tatttcgcca?aaacac

Select following primer sequence for genes of SEQ ID NO:27329:

I) forward primer (SEQ ID NO:27375)

atgaaaaatg?ccaatcatcg?attcttc

Ii) reverse primer (SEQ ID NO:27376)

ttagaagaac?agtgacggat?cgcc

Select following primer sequence for genes of SEQ ID NO:27377:

I) forward primer (SEQ ID NO:27855)

atgtctgagc?agtttttgta?tttctt

Ii) reverse primer (SEQ ID NO:27856)

ttatactttc?tctacctccg?ggcg

Select following primer sequence for genes of SEQ ID NO:27857:

I) forward primer (SEQ ID NO:28007)

atgttgtcga?gactatcttt?attgag

Ii) reverse primer (SEQ ID NO:28008)

ttaaaataga?ccttcaattt?caccgt

Select following primer sequence for genes of SEQ ID NO:28009:

I) forward primer (SEQ ID NO:28143)

atggagacca?atttttcctt?cgact

Ii) reverse primer (SEQ ID NO:28144)

ctattgaaat?accggcttca?atattt

Select following primer sequence for genes of SEQ ID NO:28145:

I) forward primer (SEQ ID NO:28177)

atgtcagcag?acgaaacgga?tgc

Ii) reverse primer (SEQ ID NO:28178)

ttatgttttt?ttgtctgctg?cagct

Select following primer sequence for genes of SEQ ID NO:28179:

I) forward primer (SEQ ID NO:28195)

atgaatgatc?ctcgtgaaat?tttagc

Ii) reverse primer (SEQ ID NO:28196)

ttatattatc?tcaagatcgc?tggca

Select following primer sequence for genes of SEQ ID NO:28197:

I) forward primer (SEQ ID NO:28199)

atgtccaact?ttaagaattt?tactttaa

Ii) reverse primer (SEQ ID NO:28200)

tcatttgttt?atcagtgtaa?caagca

Select following primer sequence for genes of SEQ ID NO:28201:

I) forward primer (SEQ ID NO:28205)

atgaatcaga?gcgatagcag?cttg

Ii) reverse primer (SEQ ID NO:28206)

tcatcttcga?agataagggg?tattc

Select following primer sequence for genes of SEQ ID NO:28207:

I) forward primer (SEQ ID NO:28209)

atggcggttg?cgatcaaaaa?gga

Ii) reverse primer (SEQ ID NO:28210)

tcaattgata?aatgtacttt?caatgatg

Select following primer sequence for genes of SEQ ID NO:28211:

I) forward primer (SEQ ID NO:28339)

atgagttctg?tcgcagaaaa?tataat

Ii) reverse primer (SEQ ID NO:28340)

ttattcgaag?acttctccag?taattg

Select following primer sequence for genes of SEQ ID NO:28341:

I) forward primer (SEQ ID NO:28361)

atgaactcta?ttttagacag?aaatgtt

Ii) reverse primer (SEQ ID NO:28362)

ttatttttgg?tcttgtttca?aagtgtc

Select following primer sequence for genes of SEQ ID NO:28363:

I) forward primer (SEQ ID NO:28367)

atgttaaaaa?gacgctctaa?tgctc

Ii) reverse primer (SEQ ID NO:28368)

tcaccaaaac?catctcctat?accaa

Select following primer sequence for genes of SEQ ID NO:28369:

I) forward primer (SEQ ID NO:28563)

atgactgaaa?gtgctataga?tgacc

Ii) reverse primer (SEQ ID NO:28564)

ctataaaatt?tgtgtatgaa?taaataaag

Select following primer sequence for genes of SEQ ID NO:98288:

I) forward primer (SEQ ID NO:98448)

atgatccgca?gtatgaccgc?ct

Ii) reverse primer (SEQ ID NO:98449)

ttattcgatg?ttctgaatct?gctc

Select following primer sequence for genes of SEQ ID NO:98533:

I) forward primer (SEQ ID NO:98701)

ttaaagcgta?tgtgtttcat?atgcc

Ii) reverse primer (SEQ ID NO:98702)

atgacagaat?tttattctga?cacaatc

Select following primer sequence for genes of SEQ ID NO:97771:

I) forward primer (SEQ ID NO:97961)

atgtctattg?tggtgaaaaa?taacatt

Ii) reverse primer (SEQ ID NO:97962)

ttattttgcc?tccgatgcca?gttct

Select following primer sequence for genes of SEQ ID NO:97966:

I) forward primer (SEQ ID NO:98282)

atgacgaatc?cgttactgac?tcc

Ii) reverse primer (SEQ ID NO:98283)

ttagccctta?atgccgtaat?gctcc

Select following primer sequence for genes of SEQ ID NO:98453:

I) forward primer (SEQ ID NO:98527)

atgacgcaga?cttccgcatt?tca

Ii) reverse primer (SEQ ID NO:98528)

ttacgccacc?gtcaactgtc?cg

[0468.0.0.0] was to [0470.0.0.0] above [0468.0.0.21] to [0470.0.0.21] saw for disclosing of [0468.0.0.21] to [0470.0.0.21] these paragraphs

The PCR product that [0470.1.21.21] utilizes the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBIFermentas) phosphorylation to produce by Pfu Turbo archaeal dna polymerase, and the clone enters in the binary vector of treated mistake.

[0471.0.21.21] sees [0471.0.0.0] for disclosing of this paragraph

[0471.1.21.21] uses Pfu Turbo archaeal dna polymerase benefit flat in the second step building-up reactions by the DNA end of the PCR product that the Herculase archaeal dna polymerase produces.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Utilize the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBI Fermentas) phosphorylation PCR product, and be cloned in the carrier of handling.

[0472.0.0.0] was to [0479.0.0.0] above [0472.0.0.21] to [0479.0.0.21] saw for disclosing of [0472.0.0.21] to [0479.0.0.21] these paragraphs

[0480.0.21.21] embodiment 11: express the generation of SEQ ID NO:14364,27329,27377,27857,28009,28145,28179,28197,28201,28207,28211,28341,28363,28369,98288,98533,97771,97966 or 98453 transgenic plant

[0482.0.0.0] was to [0513.0.0.0] above [0481.0.0.21] to [0513.0.0.21] saw for disclosing of [0481.0.0.21] to [0513.0.0.21] these paragraphs

[0514.0.21.21] alternatively, as Yaqoob M etc., J Biolumin Chemilumin.1997Jan-Feb; 12 (1): detect glycerol-3-phosphate described in the 1-5.As Foglia, T.A. etc. detect glycerine described in the 2004.Chromatographia.60:305-311.

The different plants of being analyzed the results are shown in following table 1:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??YDR065	Glycerol-3-phosphate	??GC	??1.18	??2.14
??YBR084W	Glycerine, the polarity part	??GC	??2.02	??3.53
					??YDR513W	Glycerine, the lipid part	??GC	??1.17	??1.48
??YDR513W	Glycerine, the polarity part	??GC	??1.40	??3.00
					??YGL237C	Glycerine, the lipid part	??GC	??1.23	??1.73

ORF	Metabolite	Method	Min	?Max
					?YIL150C	Glycerine, the lipid part	GC	?1.94	?3.19
?YLR082C	Glycerine, the polarity part	GC	?1.41	?2.73
					?YLR224W	Glycerine, the lipid part	GC	?1.17	?1.43
?YLR255C	Glycerine, the polarity part	GC	?1.44	?1.73
					?YMR015C	Glycerine, the lipid part	GC	?1.18	?1.23
?YOR344C	Glycerine, the lipid part	GC	?1.18	?2.04
					?YPL099C	Glycerine, the polarity part	GC	?1.55	?2.04
?YPL268W	Glycerine, the polarity part	GC	?1.38	?2.15
					?b2441	Glycerine, the polarity part	GC	?1.41	?1.81
?b3457	Glycerine, the lipid part	GC	?1.18	?1.44
					?YHR072W	Glycerine, the lipid part	GC	?1.19	?2.09
?b3644	Glycerine, the lipid part	GC	?1.17	?1.44
					?b3498	Glycerol-3-phosphate, the lipid part	GC	?1.20	?1.56
?b2710	Glycerol-3-phosphate, the lipid part	GC	?1.20	?1.95
					?b4073	Glycerol-3-phosphate, the lipid part	GC	?1.49	?2.45

[0515.0.21.21] the 2nd row have shown metabolite glycerine or the glycerol-3-phosphate of being analyzed.According to its solubleness, the free glycerol in the cell is present in polarity part (glycerine, polarity part) and measures therein.As described in top embodiment 13,, use the ester functional group of the mixture process residue of methyl alcohol and hydrochloric acid with the fracture analysis thing with nonpolar extract (=lipid part) evaporation.Will be by this reaction in conjunction with glycerine up to three ester functional groups, promptly the glycerine in the lipid is transformed into free glycerol, and as the glycerine polarity detection in the lipid part.The 4th row and the 5th row have shown the ratio of metabolite between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

[0516.0.0.0] was to [0530.0.0.0] above [0516.0.0.21] to [0530.0.0.21] saw for disclosing of [0516.0.0.21] to [0530.0.0.21] these paragraphs

[0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.21] to [0530.6.0.21] saw for disclosing of [0530.1.0.21] to [0530.6.0.21] these paragraphs

[0531.0.0.0] was to [0552.0.0.0] above [0531.0.0.21] to [0552.0.0.21] saw for disclosing of [0531.0.0.21] to [0552.0.0.21] these paragraphs

[0552.1.0.21]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF title	Metabolite	??Min	??Max
				?YIL150C	Glycerine, the lipid part	??2.12	??2.66
?YBR084W	Glycerine, the polarity part	??1.36	??1.44
				?YLR082C	Glycerine, the polarity part	??1.12	??1.35

Table 2 shows that glycerine has increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YIL150c, YBR084W or YLR082C.

In one embodiment, under the situation that yeast saccharomyces cerevisiae protein YIL150C or its homologue are enhanced as the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue in maize plant, preferably, giving fine chemicals glycerine is increased between 112% and 166%.

In one embodiment, yeast saccharomyces cerevisiae protein YBR084W or its homologue in maize plant, for example have under the situation that the activity of the protein of C1-tetrahydrofolic acid (THFA) synthase activity or its homologue is enhanced, preferably, give fine chemicals glycerine and be increased between 36% and 44%.

In one embodiment, yeast saccharomyces cerevisiae protein YLR082C or its homologue in maize plant, for example under the situation that the activity of the invalid lethal arrestin protein of Rad53 or its homologue is enhanced, preferably, give fine chemicals glycerine and be increased between 12% and 35%.

[0552.2.0.21] sees [0552.2.0.0] for disclosing of this paragraph

[0553.0.21.21]

1. produce the method for glycerol-3-phosphate or glycerine, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation glycerol-3-phosphate or glycerine.

2. produce the method for glycerol-3-phosphate or glycerine, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, and described nucleic acid molecule is given in biology or its part glycerol-3-phosphate or glycerine quantity increases;

B) contain just like Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of glycerol-3-phosphate or glycerine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of glycerol-3-phosphate or glycerine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that glycerol-3-phosphate in biology or its part or glycerine quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, the 173rd, 262-274 and 628-632 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives in biology or its part glycerol-3-phosphate or glycerine quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part glycerol-3-phosphate or glycerine quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, the 173rd, 262-274 and 628-632 capable shown in consensus sequence polypeptide and give biology or its part in glycerol-3-phosphate or glycerine quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give glycerol-3-phosphate or the increase of glycerine quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded glycerol-3-phosphate or glycerine.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or the bonded glycerol-3-phosphate or the glycerine of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, and described nucleic acid molecule is given in biology or its part glycerol-3-phosphate or glycerine quantity increases;

B) contain just like Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of glycerol-3-phosphate or glycerine quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of glycerol-3-phosphate or glycerine quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that glycerol-3-phosphate in biology or its part or glycerine quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, the 173rd, 262-274 and 628-632 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives in biology or its part glycerol-3-phosphate or glycerine quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part glycerol-3-phosphate or glycerine quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, the 173rd, 262-274 and 628-632 capable shown in consensus sequence polypeptide and give biology or its part in glycerol-3-phosphate or glycerine quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give glycerol-3-phosphate or the increase of glycerine quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding described in claim 6, thus this polypeptide by one or more amino acid be different from as Table II the 5th or 7 be listed as, the 173rd, 262-274 and 628-632 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in glycerol-3-phosphate or glycerine quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part glycerol-3-phosphate or glycerine quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps glycerol-3-phosphate or glycerine level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the glycerol-3-phosphate that will measure or glycerine level or expression of polypeptides level and the standard glycerol-3-phosphate of when described candidate compound or the sample that comprises described multiple compound lack, measuring or glycerine or expression of polypeptides level; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that glycerol-3-phosphate in plant or the microorganism or glycerine produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of glycerol-3-phosphate or glycerine quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of expression of polypeptides of glycerol-3-phosphate in biology or its part or the increase of glycerine quantity and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that glycerol-3-phosphate in the cell or glycerine produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that glycerol-3-phosphate or glycerine increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce glycerol-3-phosphate or glycerine;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) glycerol-3-phosphate or the glycerine level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give glycerol-3-phosphate or the raising of glycerine level in the host cell after it is expressed with wild-type.

20. identify and to give the method that glycerol-3-phosphate in the cell or glycerine produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that glycerol-3-phosphate in biology or its part or glycerine quantity or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce glycerol-3-phosphate or glycerine;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) glycerol-3-phosphate or the glycerine level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give glycerol-3-phosphate or the raising of glycerine level in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify the nucleic acid molecule of giving glycerol-3-phosphate or glycerine increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control glycerol-3-phosphate or glycerine level at biology.

25. agrochemicals, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of any one described method of claim 1-5, claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein fine chemicals is glycerol-3-phosphate or glycerine.

27. according to claim 10-12 any one described host cell or plant, its anti-glycerol-3-phosphate or biosynthetic weedicide of glycerine of suppressing.

28. be used to improve the method that total fat produces, it is included in to improve in biology or its part or produce expresses at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding be as polypeptide or its fragment shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable, and described nucleic acid molecule gives that total fat quantity increases in biology or its part;

B) contain just like Table I the 5th or 7 row, the 173rd, 262-274 and 628-632 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of total fat quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of total fat quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that total fat quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 7th row, the 173rd, 262-274 and 628-632 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives that total fat quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that total fat quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, the 173rd, 262-274 and 628-632 capable shown in consensus sequence polypeptide and give biology or its part in total fat quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give total fat quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

29. coding is as the purposes of polypeptide shown in Table II the 5th or 7 row, the 173rd, 262-274 and 628-632 are capable or its segmental nucleic acid molecule, wherein said nucleic acid molecule is given total fat quantity increase in biology or its part, and it is used to identify gives the nucleic acid molecule that total fat quantity increases after expression.

[0554.0.0.21] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.22] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.22] sees [0001.0.0.0] for disclosing of this paragraph.

[0002.0.22.22] lipid obviously is different from the biomolecules and the metabolite of other kind.By definition, lipid is the water-insoluble biomolecules of high dissolution in organic solvent such as chloroform.Lipid has the various biological effect: they can be used as fuel molecule, highly enriched energy storage thing, signaling molecule and film component.

The main kind of film lipid is phosphatide, glycolipid and cholesterol.Glycolipid is to contain the glycolipid class.The name of term glycolipid contains any compound that is connected to the one or more monosaccharide residues of hydrophobic part such as acylglycerol, sphingosine, ceramide (N-acyl sphingosine) or phosphoric acid isoprene by glycosidic link.

[0003.0.22.22] contains the main nonprotein composition that semi-lactosi lipid is photosynthetic membrane in plant, algae and the various bacteria.In higher plant, galactolipid comprises a high proportion of polyunsaturated fatty acid, and wherein as many as 95% may be linolenic acid (18:3 (n-3)).For example organize in rhizome or the root C at non-photosynthesis ₁₈Lipid acid is mostly usually to be saturated.

In plant, particularly in the photosynthesis tissue, the substantial part of lipid comprises that the glycosidic link by position sn-3 place is connected to 1 of carbohydrate part, 2-diacyl-sn-glycerine.Two kinds of the most general galactolipids are single galactosyl diacylglycerol and digalactosyl diacylglycerol.80% of the whole lipids of as many as is relevant with photosynthetic membrane in the plant, and single galactosyl diacylglycerol is generally believed it is in the world the abundantest film lipid.

Single galactosyl diacylglycerol is not only a plant lipid, because have been found that in its brain and nervous tissue that is present in some animal species on a small quantity.

Have been found that those phytoglycolipid main components related compound for example single and the digalactosyl diacylglycerol has 4 galactose units of as many as, perhaps one of them or manyly substituted by glucose moiety.In addition, 6-O-acyl group-single galactosyl diacylglycerol also is the component of plant tissue occasionally.

Final step betides outside the plastid and by single galactosyl diacylglycerol synthase (EC 2.4.1.46) catalysis and is undertaken in the biosynthesizing of [0004.0.22.4] single galactosyl diacylglycerol.This enzyme will be transferred to sn-1 from the UDP-semi-lactosi from the D-semi-lactosi, 2-diacylglycerol (DAG) (Joyard J.﹠amp; Douce, R.Stumpf, P.K. writes (1987) in Biochemistry of Pant (Academic, NewYork)).

Digalactosyl diacylglycerol synthase catalysis semi-lactosi to another, produces digalactosyl diacylglycerol and DAG with equimolar amount from a single galactosyl diacylglycerol molecular transfer.

Even some details are known, it is very complicated that the galactolipid biosynthesizing in the plant remains [0005.0.22.22].The galactolipid biosynthesizing relates to many approach that produce differing molecular.

Nearest studies show that the amount of lipid sulfatide sulfo-quinovose-diacylglycerol (SQDG) and digalactosyl diacylglycerol and DGDG significantly improves during phosphoric acid salt lacks (

Deng, Proc.Natl.Acad.Sci., 97,10649-10654,2000).Prescribe a time limit when phosphoric acid salt has, the phosphatide in the plant membrane reduces and is alternative by glycolipid (being SQDG and DGDG) to small part.

Except alternative lipid as phosphatide, galactolipid for the stability of photosynthesis complex body in the thylakoid also be critical (

And Benning, Trends Plant Sci.7,112-118,2002).

[0006.0.22.22] differs widely with plant, and plant contains a large amount of glycolipids (glycolipid carries sugar moieties at head group), and phosphatide is very abundant in animal and yeast.Even so, a class glycolipid of finding in Mammals is gal-cer (cerebroside), and it is ubiquity in brain and central nervous system.Cerebroside is positioned the outer side of plasma membrane, is exposed to cell surface.As if they can determine different blood groups.Blood group antigen comprise the cerebroside that is attached with a plurality of sugar.

[0007.0.22.22.] has realized that for a long time the glycolipid antigen of many complexity participates in combination on Sugar receptors and antigen only comprises single sugared head at cell surface the glycolipid, and can in film, exercise the combined action (Varki etc. of immunity, adjusting and structure, Essentials of Glycobiology.Cold Spring Harbor Laboratory Press, New York, 1999).

Sphingoglycolipid (gal-cer) has shown it is crucial activator (Zhao etc., Cancer Res.59, the 482-486 of trigger cell death; 1999), and have effect (Kakimi etc., a J.Exp.Med.192 that suppresses virus replication; 921-930,2000).

Verified recently in the film of some microorganisms, galactolipid also is responsible for preventing cell injury and is given oxygenizement and hot height resistance (Nakata, J.Biochem.127,731-737,2000).

[0008.0.22.22.] other glycosyl glyceride is as 1; 2-two-O acyl group-3-O-(D-galactopyranose base)-sn-glycerine extensively exists in the constituent of occurring in nature as the higher plant photosynthetic membrane, and is present in protokaryon blue-green algae and several other microbial cell film and in cereal grass such as wheat and the avenaceous seed.

[0009.0.22.22.] galactolipid is one of abundant more lipid of occurring in nature.The source of galactolipid is a food, some cereal (oat, wheat, barley and corn) for example, and they have just become the integral part of human diet since free record.

In addition, galactolipid comprises important lipid acid for example linolic acid, linolenic acid and other lipid acid, and they are at food and fodder industry, have extensive application at makeup and medicine field.For example, for bluish-green bacterium and marine green algae fermentation, having described gamma-linolenic acid (GLA) is galactolipid part (Cohen etc., the J.Appl.Phycol. that is restricted; (1993) 5,1,109-15; FEMS-Microbiol.Lett.; (1993) 107,2-3 163-67), this means that the concentration that increases galactolipid may be the another kind of method that increases purpose lipid acid (as linolic acid, linolenic acid, stearic acid and palmitinic acid) concentration in some production systems.

Vegetables and fruit contain galactolipid in [0010.0.22.22] humans and animals diet overwhelming majority, and their degradation production has been represented semi-lactosi and the important diet source of polyunsaturated fatty acid.

[0011.0.22.22] is owing to semi-lactosi and contain the positive characteristics of lipid semi-lactosi and useful physiological role, still needs to produce in a large number quality and those fully definite compounds of component.

[0012.0.22.22.] therefore still expects to produce galactolipid with the ratio of determining in microorganism or plant.This method should not rely on phosphatic operability, and particularly phosphoric acid salt lacks.A kind of method that improves biosynthesizing throughput is to use recombinant DNA technology.This type production allows the most suitable and the most effective quality, quantity and the screening of producing biology of control.The latter is particularly important for commercial production economics, and also is favourable for the human consumer therefore.

More already used year of [0013.0.22.22] recombinant DNA technology method is with by increasing different biosynthesis genes and study the production that its influence to fine chemicals production improves fine chemicals in microorganism and the plant.

New enzymic activity or direct or indirect instrumentality still constantly need be provided, and the alternative approach that therefore need have advantageous feature is used for containing semi-lactosi and lipid acid as genetically modified organism with definite ratio production biological, preferably galactose fat or cerebroside, compound or its precursor.

[0014.0.22.22] therefore in the first embodiment, the present invention relates to produce the method for fine chemicals, and fine chemicals is fat, preferred glycolipid, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside thus.

Therefore, in the present invention, term " fine chemicals " refers to " fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside " as used herein.

In addition, as used herein term " fine chemicals " also refer to comprise fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic fine chemicals.

[0015.0.22.22] in one embodiment, term " fine chemicals " meaning is meant fat, preferred glycolipid, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.

In whole specification sheets, term " fine chemicals " meaning be meant free form or be bonded to the fat of compound form, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside, for example sulfonate.In preferred embodiments, term " fine chemicals " meaning is meant free form or its salt or is bonded to the galactolipid of sulphate form.

[0015.1.22.22] to fat of the present invention, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside Determination on content are to measure semi-lactosi pyranoside, preferable methyl semi-lactosi pyranoside.If sample is preparation as be shown in the examples and mensuration, then this compound is indication fat of the present invention, preferred glycolipid, comprises the glycolipid of semi-lactosi, the more preferably analyte that exists of galactolipid and/or cerebroside.

[0016.0.22.22] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: YLR224W, YLR255C, YER173W, b2699, b3129 and/or YHR072W protein; With

(b) to produce fine chemicals in allowing described biology be fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably make biological growth under galactolipid and/or the cerebronic condition.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in activity of proteins, perhaps have by Table I the 5th or 7 row and/or the 633rd and 634 the row shown in nucleic acid molecule encoded polypeptide sequence and

(b) to produce fine chemicals in allowing described biology be fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably make biological growth under galactolipid and/or the cerebronic condition.

Above [0017.0.0.22] and [0018.0.0.22] sees for disclosing of [0017.0.0.22] and [0018.0.0.22] these paragraphs [0017.0.0.0] and [0018.0.0.0]

The method that [0019.0.22.22] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in capable and/or the 633rd and 634 row of 186-189 or by as Table I the 5th or 7 be listed as, the activity of proteins of the coded protein active of nucleic acid molecule is carried out above-mentioned modification shown in capable and/or the 633rd and 634 row of 186-189.

[0020.0.22.22] is surprisingly found out that, the e. coli k12 protein b2699 shown in yeast saccharomyces cerevisiae protein YLR224W, YLR255C, YER173W and/or YHR072W shown in Table II the 3rd or 5 row, 186-188 and 634 row and/or Table I the 3rd or 5 row, the 189th and 633 row and/or b3129 transgene expression in Arabidopis thaliana fat of giving the plant that transforms, preferably glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or the increase of cerebroside content.Therefore, in one embodiment, described protein or its homologue (for example Table II the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in) be used to produce fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.

[0021.0.0.22] sees [0021.0.0.0] for disclosing of this paragraph

The sequence of [0022.0.22.22] yeast saccharomyces cerevisiae YLR224W has been published in Johnston etc., Nature 387 (6632 supplementary issue), 87-90 (1997) and Goffeau etc., Science 274 (5287), 546-547,1996, and its cytoactive is not still characterized.As if it have the activity of yeast saccharomyces cerevisiae putative protein matter YLR224w superfamily.Therefore, in one embodiment, the inventive method comprises having the active gene product of putative protein matter YLR224w superfamily, preferably have the protein of yeast saccharomyces cerevisiae YLR224w protein active or a purposes of its homologue from yeast saccharomyces cerevisiae as shown here, it is used for producing fine chemicals in biological or its part as described, promptly fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside, particularly free or combining form quantity.In a further embodiment, the YLR224w protein expression is enhanced with another expression of gene of semi-lactosi approach, and preferably the expression of gene that participates in comprising protein that the glycolipid of semi-lactosi produces or coding semi-lactosi transporter protein or compound (its as each fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic storage pond) with coding is enhanced.In one embodiment, in the methods of the invention, yeast saccharomyces cerevisiae putative protein matter YLR224w superfamily protein, preferred YLR224w activity of proteins are enhanced or produce, and for example the activity from the putative protein matter YLR224w superfamily protein of yeast saccharomyces cerevisiae, preferred YLR224w protein or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YLR255C is committed to EMBL DataLibrary by people such as Johnson, and (February nineteen ninety-five), and its activity do not characterize yet, but preferably have yeast belong putative protein matter YLR255c superfamily activity.Therefore, in one embodiment, the inventive method comprises as shown here having the active gene product of yeast belong putative protein matter YLR255c superfamily, preferably have the gene product of yeast saccharomyces cerevisiae YLR255C protein active or a purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, promptly fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside, particularly increase the quantity of free or combining form.In a further embodiment, the YLR255C protein expression is enhanced with another expression of gene of semi-lactosi approach, and preferably the expression of gene that participates in protein that glycolipid produces or coding semi-lactosi transporter protein or compound (its as each fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic storage pond) with coding is enhanced.In one embodiment, in the methods of the invention, yeast belong putative protein matter YLR255c superfamily protein, preferred YLR255C activity of proteins is enhanced or produces, for example from the putative protein matter YLR255c superfamily protein of yeast saccharomyces cerevisiae, the activity of preferred YLR255C protein or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YER173W has been published in Dietrich etc., Nature 387 (6632 supplementary issue), 78-81,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be to participate in dna damage and reduction division pachytene stage check position activatory check position protein.Therefore, in one embodiment, the inventive method comprises as shown here the have dna damage of participation and the gene product of reduction division pachytene stage check position activatory check position protein active or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, promptly fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside, particularly increase the quantity of free or combining form.In a further embodiment, the YER173w protein expression is enhanced with another expression of gene of semi-lactosi approach, preferably with coding participate in fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably the proteinic expression of gene that produces of galactolipid and/or cerebroside is enhanced.In one embodiment, in the methods of the invention, participate in dna damage and reduction division pachytene stage check position activatory check position activity of proteins and be enhanced or produce, for example be enhanced or produce from the participation dna damage of yeast saccharomyces cerevisiae and the activity of reduction division pachytene stage check position activatory check position protein or its homologue.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be DNA chain exchange and the recombinant protein with recombinant protein recA superfamily proteolytic enzyme and nuclease.Therefore, in one embodiment, the inventive method comprises as shown here recombinating and the DNA repairing activity from the colibacillary DNA of having, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, the purposes of recombinant protein recA superfamily or its homologue particularly, it is used for producing each fine chemicals in biological or its part as described, be fat, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably galactolipid and/or cerebroside, the quantity of preferably free or combining form.In a further embodiment, the b2699 protein expression is enhanced with another expression of gene of semi-lactosi approach, preferably with coding participate in fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably the proteinic expression of gene that produces of galactolipid and/or cerebroside is enhanced.In one embodiment, in the methods of the invention, described activity for example proteolytic enzyme and nuclease, particularly have the DNA chain exchange of proteolytic enzyme and nuclease and activity, the particularly activity of recombinant protein recA superfamily of recombinant protein and be enhanced or produce, for example the activity from colibacillary these protein or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YHR072W (accession number NP_011939) has been published in Goffeau etc., Science 274 (5287), Science 265 (5181) such as 546-547 (1996) and Johnston, 2077-2082 (1994), and it is 2 that its activity is defined as, 3-epoxy squalene-lanosterol cyclase protein matter.Therefore, in one embodiment, the inventive method comprises the protein of the stress-induced that does not characterize from yeast saccharomyces cerevisiae as shown here or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside, the quantity of preferably free or combining form.In one embodiment, in the methods of the invention, 2, the activity of 3-epoxy squalene-lanosterol cyclase protein matter is enhanced or produces, for example from 2 of yeast saccharomyces cerevisiae, the activity of 3-epoxy squalene-lanosterol cyclase protein matter or its homologue is enhanced or produces.

The sequence of e. coli k12 b3129 (accession number NP_417598) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be " the proteolytic enzyme of supposition; HtrA repressor ".Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary " proteolytic enzyme of supposition; The htrA repressor " or the purposes of its homologue; it is used for producing fine chemicals in biological or its part as described; be fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside, preferably free or combining form quantity." the proteolytic enzyme of supposition in the methods of the invention in one embodiment; The htrA repressor " activity be enhanced or produce, for example from the colibacillary " proteolytic enzyme of supposition; The htrA repressor " or its homologue.

The homologous compound (=homologue) of [0023.0.22.22] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given fine chemicals quantity or content and increase.In addition, in the present invention, the sequence that term " homologous compound " relates to described herein or listed described all expressed sequences of biology has the sequence of the biology of highest serial homology.Yet, it be known to those skilled in the art that active and (if known words) that homologous compound preferably has a described increase fine chemicals in biology with protein shown at least a Table I the 3rd row, 186-188 and 634 row, for example have by the protein that contains the coded peptide sequence of nucleotide sequence of sequence shown in Table I the 5th or 7 row, 186-188 and 634 row and have identical biological function or activity.

In one embodiment, any one homologue of polypeptide shown in Table II 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in the biology) and from Eukaryotic homologue.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 189th and 633 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of bacterium.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of fungi.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 189th and 633 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of Proteobacteria.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of Ascomycota.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 189th and 633 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of γ-distortion Gammaproteobacteria.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from saccharomycetic homologue.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 189th and 633 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from enterobacteria purpose homologue.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of yeast guiding principle.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 189th and 633 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of enterobacteriaceae.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of Saccharomycetes.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 189th and 633 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of Escherichia.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of Saccharomycetaceae.

In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, 186-188 and 634 row is to have same or similar activity (particularly, active raising give fine chemicals content increases in biology or its part) and from the homologue of yeast guiding principle

[0023.1.22.22] Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in the homologue of polypeptide can be by Table I the 7th row, 186-189 is capable and/or the 633rd and/or 634 row shown in the polypeptide of nucleic acid molecule encoding or Table II the 7th row, 186-189 capable shown in polypeptide.

Table II the 3rd row, 186-189 is capable and/or the 633rd and/or 634 row shown in the homologue of polypeptide can be by Table I the 7th row, 186-189 is capable and/or the 633rd and/or 634 row shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, 186-189 is capable and/or the 633rd and/or 634 row shown in polypeptide.

[0024.0.0.22] sees [0024.0.0.0] for disclosing of this paragraph

[0025.0.22.22] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause fat, preferred glycolipid in biology or its part, the preferred described biomass cells, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside level increase, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, 186-189 capable and/or the 633rd and 634 the row shown in any one proteinic biology or the enzyme activity, if promptly with Table II the 3rd row, yeast saccharomyces cerevisiae protein shown in 186-188 and/or 634 row or Table II the 3rd row, shown in the 189th and/or 633 row in the e. coli k12 protein any one compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

Above [0025.1.0.22] and [0025.2.0.22] sees for disclosing of [0025.1.0.22] and [0025.2.0.22] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.22] to [0033.0.0.22] saw for disclosing of [0026.0.0.22] to [0033.0.0.22] these paragraphs

[0034.0.22.22] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, its have Table II the 3rd row, 186-189 is capable and/or the 633rd and/or 634 row shown in protein or by Table I the 5th row, 186-189 is capable and/or the 633rd and/or 634 row shown in the protein of nucleic acid molecule encoding or its homologue (as Table I the 7th row, 186-189 is capable and/or the 633rd and/or 634 row shown in homologue) active protein expression level or active aspect difference, and its difference aspect biological chemistry or genetics reason.Therefore it shows the fine chemicals quantity that increases.

[0035.0.0.0] was to [0036.0.0.0] above [0035.0.0.22] and [0036.0.0.22] saw for disclosing of [0035.0.0.22] and [0036.0.0.22] these paragraphs

There is a series of mechanism in [0037.0.22.22], and the modification by these machine-made egg white matters (polypeptide for example of the present invention) can directly or indirectly influence fat, preferred glycolipid, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic output, production and/or production efficiency.

[0038.0.0.0] was to [0044.0.0.0] above [0038.0.0.22] to [0044.0.0.22] saw for disclosing of [0038.0.0.22] to [0044.0.0.22] these paragraphs

[0045.0.22.22] in one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR224W or its homologue (for example shown in Table II the 5th or 7 row, the 186th row) is enhanced, preferably, give fine chemicals be increased in 15% and 46% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR255C or its homologue (for example shown in Table II the 5th or 7 row, the 187th row) is enhanced, preferably, give in one embodiment fine chemicals be increased in 16% and 30% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER173w or its homologue (for example shown in Table II the 5th or 7 row, the 188th row), for example participate under the situation that dna damage and reduction division pachytene stage check position activatory check position activity of proteins be enhanced, preferably, give fine chemicals be increased in 20% and 70% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2699 or its homologue (for example shown in Table II the 5th or 7 row, the 189th row) is enhanced, for example have under the situation that the activity of the DNA chain exchange of recombinant protein recA superfamily proteolytic enzyme and nuclease and recombinant protein or its homologue is enhanced, preferably, give fine chemicals be increased in 15% and 64% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b3129 or its homologue (for example shown in Table II the 5th or 7 row, the 633rd row) is enhanced, " the proteolytic enzyme of supposition for example; The htrA repressor " or the activity of its homologue situation about being enhanced under, preferably, give fine chemicals be increased in 16% and 32% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YHR072W or its homologue (for example shown in Table II the 5th or 7 row, the 634th row) is enhanced, for example 2, under the situation that the activity of 3-epoxy squalene-lanosterol cyclase protein matter or its homologue is enhanced, preferably, give fine chemicals be increased in 17% and 35% or more between.

[0046.0.22.22] in one embodiment, yeast saccharomyces cerevisiae protein YLR224W or its homologue, for example the activity of YLR224W protein (for example shown in Table II the 5th or 7 row, the 186th row) is given each fine chemicals and other lipid, preferred glycolipid, sphingolipid and/or galactolipid or their precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YLR255C or its homologue, for example the activity of YLR255C protein (for example shown in Table II the 5th or 7 row, the 187th row) is given each fine chemicals and other lipid, preferred glycolipid, sphingolipid and/or galactolipid or their precursor increases.

In one embodiment, yeast saccharomyces cerevisiae protein YER173W or its homologue, the activity that for example participates in dna damage and reduction division pachytene stage check position activatory check position protein (for example shown in Table II the 5th or 7 row, the 188th row) gives each fine chemicals and other lipid, preferred glycolipid, sphingolipid and/or galactolipid or their precursor increases.

In one embodiment, e. coli k12 protein b2699 or its homologue, the activity that for example has the DNA chain exchange of recombinant protein recA superfamily proteolytic enzyme and nuclease and recombinant protein (for example shown in Table II the 5th or 7 row, the 189th row) is given each fine chemicals and other lipid, preferred glycolipid, sphingolipid and/or galactolipid, compound or the increase of their precursor.

In one embodiment, e. coli k12 protein b3129 or its homologue for example have " the proteolytic enzyme of supposition; The htrA repressor " activity of active protein (for example shown in Table II the 5th or 7 row, the 633rd row) gives each fine chemicals and other lipid, preferred glycolipid, sphingolipid and/or galactolipid, compound or their precursor and increases.

In one embodiment, at yeast saccharomyces cerevisiae protein YHR072W or its homologue, for example 2, the activity of 3-epoxy squalene-lanosterol cyclase protein matter (for example shown in Table II the 5th or 7 row, the 634th row) is given each fine chemicals and other lipid, preferred glycolipid, sphingolipid and/or galactolipid or their precursor to be increased.

Above [0047.0.0.22] and [0048.0.0.22] sees for disclosing of [0047.0.0.22] and [0048.0.0.22] these paragraphs [0047.0.0.0] and [0048.0.0.0]

[0049.0.22.22] has to give and improves fine chemicals quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide of the present invention comprises as Table IV the 7th row, the sequence of consensus sequence shown in capable and/or the 633rd and/or 634 row of 186-189 or as Table II the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in capable and/or the 633rd and/or 634 row of 186-189, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in capable and/or the 633rd and/or 634 row of 186-189) sequence of coded polypeptide, and have the activity described in the literary composition.

[0050.0.22.22] for the purposes of the present invention, term " fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside " also comprises and contains 3,4 or single semi-lactosi diacylglycerol and/or the digalactosyl diacylglycerol and/or the galactolipid of more a plurality of galactose units.

[0051.0.22.22] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic composition.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of lipid acid.

[0052.0.0.22] sees [0052.0.0.0] for disclosing of this paragraph

[0053.0.22.22] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein give the protein of nucleic acid molecule encoding of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide) express and increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA give the coded protein of nucleic acid molecule of the present invention (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide) express and improve;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, by the coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide) express and improve;

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide);

(h) by add positive Expression element or remove negative Expression element improve code book invention polypeptide (for example have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) (heat shock protein of the present invention) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.22.22] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improve coded protein or have as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide) give each fine chemicals after active expression of polypeptides or the activity and increase.

[0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.22] to [0067.0.0.22] saw for disclosing of [0055.0.0.22] to [0067.0.0.22] these paragraphs

[0068.0.22.22] with can not be to fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic generation cause the mode of disadvantageous effect to introduce sudden change.

The adjusting of [0069.0.22.22] gene or its gene product is subjected to less influence and is interpreted as enzymic activity is regulated decline, causes the specific activity of gene or its product or the raising of cytoactive.The activity that the raising of enzymic activity can be regarded as enzyme improves at least 10% with initial biophase ratio, advantageously is at least 20%, 30% or 40%, particularly advantageously is at least 50%, 60% or 70%.This cause required comprise fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic compound productivity improve.

[0070.0.22.22] imports biology separately or with other assortment of genes owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, the for example favourable fat that comprises, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably galactolipid and/or cerebronic composition for example contain high level (from the physiology of nutrition angle) fat, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably galactolipid and/or cerebronic composition.

[0071.0.0.22] sees [0071.0.0.0] for disclosing of this paragraph

[0072.0.22.22] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds except fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably also have galacturonic acid, the oligosaccharides that comprises semi-lactosi or galacturonic acid and/or polysaccharide, GalN, UDP-semi-lactosi, psychosine, UDP-N-acetylgalactosamine, UDP-galacturonic acid, other glycoprotein, Sphingolipids,sialo, mucoprotein, blood-group substance and/or ceramide galactolipid and/or the cerebroside compound.

[0073.0.22.22] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or its homologue (for example Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation, reclaim (randomly separating) by the free and/or bonded fine chemicals of biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis and optional other is free and/or bonded fat, preferably glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.

[0074.0.22.22] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or bonded fine chemicals or free and bonded fine chemicals, can also choose generations wantonly, reclaim and separate (if expectation) other dissociate and/or bonded comprise fat, preferably glycolipid, comprise the glycolipid of semi-lactosi, the more preferably compound of galactolipid and/or cerebroside semi-lactosi.

[0075.0.0.0] was to [0077.0.0.0] above [0075.0.0.22] to [0077.0.0.22] saw for disclosing of [0075.0.0.22] to [0077.0.0.22] these paragraphs

[0078.0.22.22] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with fine chemicals directly processing enter in food or food additive or the animal-feed or be used for other purposes, for example be used for pharmaceutical composition.

[0079.0.0.0] was to [0084.0.0.0] above [0079.0.0.22] to [0084.0.0.22] saw for disclosing of [0079.0.0.22] to [0084.0.0.22] these paragraphs

[0085.0.22.22] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd genetic regulatory element that effectively is connected with nucleotide sequence or derivatives thereof shown in 634 row, promotor for example, perhaps (c) (a) with (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

Above [0086.0.0.22] and [0087.0.0.22] sees for disclosing of [0086.0.0.22] and [0087.0.0.22] these paragraphs [0086.0.0.0] and [0087.0.0.0]

[0088.0.22.22] is in an advantageous embodiment of the present invention, and be biological for because expressed nucleic acid molecule of the present invention has advantageously been modified fat, preferred glycolipid, comprised the glycolipid of semi-lactosi, the more preferably plant form of galactolipid and/or cerebroside content.Because the nutritive value that for example is used for the plant of animal depends on above-mentioned lipid and as the big scale of construction of the glycolipid that comprises semi-lactosi in fodder energy source, so this is very important for the plant breeder.After improving or having produced the activity of aforementioned polypeptides, perhaps produce or improved after nucleic acid molecule of the present invention or the polypeptide expression, thereby the transgenic plant that produced grow on the nutritional medium/nutritional medium in or results in the soil and subsequently.

[0088.1.0.22] sees [0088.1.0.0] for disclosing of this paragraph

[0089.0.0.0] was to [0094.0.0.0] above [0089.0.0.22] to [0094.0.0.22] saw for disclosing of [0089.0.0.22] to [0094.0.0.22] these paragraphs

[0095.0.22.22] advantageously, by method of the present invention in genetically modified organism, increase fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside pond to be to separate a large amount of pure basically fine chemicals and/or to obtain the resistance of the raising of biology and abiotic stress and obtain high yield.

[0096.0.22.22] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide (for example lipid acid transporter albumen) or compound (as lipid required in the biology or comprise the pond of the compound of semi-lactosi) and combines and can be used for producing fine chemicals in another embodiment preferred of the present invention.

[0097.0.0.22] sees [0097.0.0.0] for disclosing of this paragraph

[0098.0.22.22] in preferred embodiments, fine chemicals (fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside) be produce according to the present invention and carry out isolating where necessary.By the inventive method produce other lipid, lipid acid and/or oligosaccharides and/or its mixture or with the mixture of other lipid acid be favourable.

[0099.0.22.22] for microbial fermentation, aforementioned fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside can be accumulated in substratum and/or the cell.If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition of fine chemicals of the present invention.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.22.22] comprises the transgenic plant that the method according to this invention synthetic comprises lipid and can advantageously directly put on market, and do not need institute's synthetic oils, lipid or lipid acid are separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo.

Yet, the fine chemicals that produces according to the inventive method can also from biological, advantageously from plant with the isolated in form of its lipid, oligosaccharides and/or free galactolipid.Yet the fine chemicals that the method according to this invention produces can also be with the form of the form of its oils, fat, lipid and/or free fatty acids from biology, advantageously separate from plant.Can obtain the galactolipid that produces by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part, preferred plant seed.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will be pretreated seed or plant squeezing have been carried out or with solvent extraction or hydrolysis or extract under acidity or alkaline condition by enzymatic breaking in this mode.The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.In this mode, can separate more than 96% of compound that the inventive method produces.After this, resulting product is further processed, i.e. refining.If expectation can water thoroughly be washed products therefrom to remove residual acid or alkali in the product, and is dry then.Be to remove residual pigment in the product, can use Fuller's earth (filler ' s earth) or activated carbon bleaching.At last under vacuum vapor distillation with the product deodorizing.These chemical pure fat, preferred glycolipid or comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside composition can advantageously be applied to grocery trade, cosmetic industry, particularly pharmacy industry.

[0101.0.22.22] sees [0101.0.0.0] for disclosing of this paragraph

[0102.0.22.22] for example, fat, preferred glycolipid or comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside can separate advantageously and detect by carry out GC after suitable specimen preparation.By using standard method of analysis GC, GC-MS or TLC can clearly detect fat, preferred glycolipid or comprising the glycolipid of semi-lactosi, the more preferably existence of galactolipid and/or cerebroside product to the biology analysis of recombinating, described analytical procedure Christie and reference wherein are described (1997, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric method], Lipide33:343-353).By ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material fragmentation to be analyzed.After the fragmentation, must material is centrifugal.With pellet resuspended in distilled water, in 100 ℃ of heating 10 minutes, cooled on ice is also centrifugal, then in containing the 0.5M sulfuric acid (in methyl alcohol) of 2% Propanal dimethyl acetal, extracted 1 hour in 90 ℃, this causes producing the oil and the fat compound of hydrolysis, and this can obtain transmethylase fat (fatty acid methyl ester and as each complement (complement) of glycerine, carbohydrate, phosphoric acid).In sherwood oil, extract these fatty acid methyl esters and finally use capillary column (Chrompack, the WCOT fused silica, CP-Wax-52CB, 25 μ m, 0.32mm) gradient temperature between 170 ℃ and 240 ℃ 20 minutes and 240 ℃ carried out the GC analysis in following 5 minutes.The identity of resulting fatty acid methyl ester must be used and can define from the standard that commercial source (being Sigma) obtains.

[0103.0.22.22] in preferred embodiments, the present invention relates to produce the method for fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide is Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in; Described nucleic acid molecule is given fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use as Table III the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in the primer nucleic acid molecule that amplifier nucleic acid molecule obtains from cDNA library or genomic library, and give the increase of fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprises Table IV the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in consensus sequence and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.22.22] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in polypeptide of sequence.

[00103.2.22.22] in one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the inventive method nucleic acid molecule used therefor not by Table I B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in sequence form.In one embodiment, the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule coding schedule IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in polypeptide of sequence.

[0104.0.22.22] in one embodiment, nucleic acid molecule that uses in the inventive method and Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the sequence difference be one or more Nucleotide, perhaps not by Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, 186-189 is capable, preferred Table I A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 186-189 is capable, preferred Table II A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide of sequence.

[0105.0.0.0] was to [0107.0.0.0] above [0105.0.0.22] to [0107.0.0.22] saw for disclosing of [0105.0.0.22] to [0107.0.0.22] these paragraphs

Advantageously improved in [0108.0.22.22] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in capable and/or the 633rd and 634 row of 186-189, from Table II the 5th or 7 row, aminoacid sequence shown in capable and/or the 633rd and 634 row of 186-189 is derived or from containing Table IV the 7th row, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in capable and/or the 633rd and 634 row of 186-189, perhaps its coding has as Table I the 3rd, 5 or 7 row, the enzymic activity of polypeptide or bioactive polypeptide shown in capable and/or the 633rd and 634 row of 186-189 or for example give derivative or the homologue that each fine chemicals increases behind its expression or active the increasing.

[0109.0.0.22] sees [0109.0.0.0] for disclosing of this paragraph

[0110.0.0.22] helps the inventive method and coding has the polypeptide of the used polypeptide active of the used or of the present invention process of the inventive method (for example as Table II the 5th or 7 row, protein shown in capable and/or the 633rd and 634 row of 186-189 or by as Table I the 5th or 7 be listed as, the protein of nucleic acid molecule encoding or its homologue are (as Table II the 5th or 7 row shown in capable and/or the 633rd and 634 row of 186-189, shown in capable and/or the 633rd and 634 row of 186-189)) nucleic acid molecule can from generally open database, determine.

[0111.0.0.22] sees [0111.0.0.0] for disclosing of this paragraph

The nucleic acid molecule that uses in [0112.0.22.22] the inventive method is the isolated nucleic acid sequences form, its coding have as Table I the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide active polypeptide or have as Table II the 5th and 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the polypeptide of peptide sequence, and give each fine chemicals level and increase.

Above [0113.0.0.22] to [0120.0.0.22] sees for disclosing of [0113.0.0.22] to [0120.0.0.22] these paragraphs [0113.0.0.0] and [0120.0.0.0]

[0121.0.22.22] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in peptide sequence or its functional homologue as herein described the difference of one or more amino acid moleculars is arranged, described artificial sequence is preferably given aforementioned activity, and promptly giving the fine chemicals level after improving its activity increases.

Above [0122.0.0.22] to [0127.0.0.22] sees for disclosing of [0122.0.0.22] to [0127.0.0.22] these paragraphs [0122.0.0.0] and [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.22.22] polymerase chain reaction (PCR) amplification is used (for example as Table III the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in primer to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence or from as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence deutero-sequence produce.

[0129.0.22.22] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).The conservative region of polypeptide of the present invention is pointed out in the comparison shown in the figure.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in consensus sequence from described comparison.

[0130.0.0.0] was to [0138.0.0.0] above [0130.0.0.22] to [0138.0.0.22] saw for disclosing of [0130.0.0.22] to [0138.0.0.22] these paragraphs

[0139.0.22.22] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence hybridization, and coding is expressed and is had the lipid of increasing, preferred glycolipid, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or the active peptide of cerebroside.

[0140.0.0.0] was to [0146.0.0.0] above [0140.0.0.22] to [0146.0.0.22] saw for disclosing of [0140.0.0.22] to [0146.0.0.22] these paragraphs

[0147.0.22.22] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.22.22] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, capable and/or the 633rd and 634 row of 186-189, preferred Table I B the 5th or 7 row, nucleotide sequence or its portion homologous are at least about 30% shown in capable and/or the 633rd and 634 row of 186-189,35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly having after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue increases lipid, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably galactolipid and/or cerebronic activity.

[0149.0.22.22] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I the 5th or 7 row, capable and/or the 633rd and 634 row of 186-189, preferred Table I B the 5th or 7 row, one of nucleotide sequence or the hybridization of its part shown in capable and/or the 633rd and 634 row of 186-189, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and (as gives for example fat of each fine chemicals, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably galactolipid and/or cerebroside increase) and randomly as Table II the 5th row, capable and/or the 633rd and 634 row of 186-189, preferred Table I B the 5th or 7 row, the protein of protein active shown in capable and/or the 633rd and 634 row of 186-189.

[00149.1.22.22] randomly, with Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the nucleotide sequence of one of nucleotide sequence hybridization have another or various active, described activity for as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in protein be known activity or be used for these protein of note.

[0150.0.22.22] in addition, nucleic acid molecule of the present invention can only contain Table I the 5th or 7 row, capable and/or the 633rd and 634 row of 186-189, preferred Table I B the 5th or 7 row, the part of the coding region of one of nucleotide sequence shown in capable and/or the 633rd and 634 row of 186-189, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give lipid, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably galactolipid and/or cerebroside increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in one of sequence sense strand, Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the antisense sequences of one of sequence or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.

Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, 186-189 capable shown in the right PCR of primer will produce as Table I the 5th or 7 be listed as, 186-189 capable shown in the fragment of polynucleotide sequence.Preferably Table I B the 5th or 7 is listed as, 186-189 is capable and/or the 633rd and 634 row.

[0151.0.0.22] sees [0151.0.0.0] for disclosing of this paragraph

[0152.0.22.22] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in the abundant homology of aminoacid sequence, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or raising lipid as be shown in the examples, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably activity of galactolipid and/or cerebroside level.

[0153.0.22.22] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprise with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in aminoacid sequence minimal number amino-acid residue identical or of equal value (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in protein or its part have Table II the 3rd row for example as herein described, 186-189 is capable and/or the 633rd and 634 row shown in the activity of polypeptide.

[0154.0.22.22] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the complete amino acid sequence homology be at least about 30%, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

Above [0155.0.0.22] and [0156.0.0.22] sees for disclosing of [0155.0.0.22] and [0156.0.0.22] these paragraphs [0155.0.0.0] and [0156.0.0.0]

[0157.0.22.22] the present invention relates in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, one of nucleotide sequence (with its part) shown in capable and/or the 633rd and 634 row of 186-189 and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 5th or 7 row, polypeptide of sequence shown in capable and/or the 633rd and 634 row of 186-189 or as Table II the 5th or 7 be listed as, the nucleic acid molecule of polypeptide or its function homologue shown in capable and/or the 633rd and 634 row of 186-189.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment as Table IV the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in consensus sequence or as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in consensus sequence or as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet, in a preferred embodiment, nucleic acid molecule of the present invention do not comprise as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I A the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence.Preferably, nucleic acid molecule of the present invention be functional homologous compound or with Table I B the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in nucleic acid molecule identical.

[0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.22] to [0160.0.0.22] saw for disclosing of [0158.0.0.22] to [0160.0.0.22] these paragraphs

[0161.0.22.22] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.22] sees [0162.0.0.0] for disclosing of this paragraph

[0163.0.22.22] preferably, under stringent condition with Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.22] sees [0164.0.0.0] for disclosing of this paragraph

[0165.0.22.22] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

Above [0166.0.0.22] and [0167.0.0.22] sees for disclosing of [0166.0.0.22] and [0167.0.0.22] these paragraphs [0166.0.0.0] and [0167.0.0.0]

[0168.0.22.22] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in aminoacid sequence at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 186-189 capable and/or the 633rd with 634 the row shown in sequence identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, one of sequence shown in 186-189 is capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 186-189 capable and/or the 633rd and 634 the row shown in sequence at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 186-189 capable and/or the 633rd and 634 the row shown in sequence at least about 96%, 97%, 98% or 99% is identical.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in the contained sequence of sequence different, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in aminoacid sequence at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, capable and/or the 633rd and 634 row of 186-189, preferred Table II B the 7th row, 186-189 capable and/or the 633rd with 634 the row shown in sequence identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, capable and/or the 633rd and 634 row of 186-189, preferred Table II B the 7th row, 186-189 is capable and/or the 633rd identical at least about 70% with one of the sequence shown in 634 row, even more preferably with as Table II the 5th or 7 be listed as, capable and/or the 633rd and 634 row of 186-189, preferred Table II B the 7th row, 186-189 capable and/or the 633rd and 634 the row shown in sequence at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, capable and/or the 633rd and 634 row of 186-189, preferred Table II B the 7th row, 186-189 capable and/or the 633rd and 634 the row shown in sequence at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.0] was to [0172.0.0.0] above [0169.0.0.22] to [0172.0.0.22] saw for disclosing of [0169.0.0.22] to [0172.0.0.22] these paragraphs

[0173.0.22.22] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:14358 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:14358 sequence before use.

[0174.0.0.22] sees [0174.0.0.0] for disclosing of this paragraph

[0175.0.22.22] for example, the sequence that has 80% homology at protein level and SEQ ID NO:14355 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:14355 sequence.

[0176.0.22.22] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtains shown in capable and/or the 633rd and 634 row of 186-189 be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in capable and/or the 633rd and 634 row of 186-189,35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide has essentially identical character and discerns shown in capable and/or the 633rd and 634 row of 186-189.

[0177.0.22.22] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, capable and/or the 633rd and 634 row of 186-189, capable and/or the 633rd and 634 row of 186-189, the functional equivalent that nucleotide sequence obtains shown in capable and/or the 633rd and 634 row of preferred Table I B 186-189 be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in capable and/or the 633rd and 634 row of 186-189,35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, capable and/or the 633rd and 634 row of 186-189, the polypeptide of the essentially identical character of polypeptide shown in capable and/or the 633rd and 634 row of preferred Table II B 186-189.

[0178.0.0.22] sees [0178.0.0.0] for disclosing of this paragraph

[0179.0.22.22] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, capable and/or the 633rd and 634 row of 186-189, shown in capable and/or the 633rd and 634 row of preferred Table I B 186-189) in introduce the replacement of one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, capable and/or the 633rd and 634 row of 186-189, the nucleic acid molecule of the homologue of protein sequence shown in capable and/or the 633rd and 634 row of preferred Table II B 186-189.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B 186-189 is capable and/or the 633rd and 634 row shown in introduce sudden change in the encoding sequence.

[0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.22] to [0183.0.0.22] saw for disclosing of [0180.0.0.22] to [0183.0.0.22] these paragraphs

[0184.0.22.22] is employed to have as Table I the 5th or 7 row, capable and/or the 633rd and 634 row of 186-189, the nucleotide sequence homologous compound of sequence shown in capable and/or the 633rd and 634 row of preferred Table I B 186-189, perhaps come Table II the 5th or 7 row freely, capable and/or the 633rd and 634 row of 186-189, the homologous compound of the nucleotide sequence of sequence shown in capable and/or the 633rd and 634 row of preferred Table II B 186-189 also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B 186-189 is capable and/or the 633rd and 634 row shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.22.22] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B 186-189 are capable and/or the 633rd and 634 row shown in sequence.In one embodiment, the preferred nucleic acid molecule comprise the least possible, as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B 186-189 is capable and/or the 633rd and 634 row shown in arbitrary sequence in other Nucleotide of not showing.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B 186-189 are capable and/or the 633rd and 634 row shown in sequence identical.

The employed one or more nucleic acid molecule encodings of [0186.0.22.22] also preferred the inventive method comprise as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B 186-189 are capable and/or the 633rd and 634 row shown in polypeptide of sequence.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B 186-189 are capable and/or the 633rd and 634 row shown in sequence identical.

[0187.0.22.22] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprise as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B 186-189 are capable and/or the 633rd and 634 row shown in polypeptide of sequence and contain and be less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, in the method employed nucleic acid molecule and coding as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B 186-189 is capable and/or the 633rd and 634 row shown in the encoding sequence of sequence identical.

[0188.0.22.22] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, capable and/or the 633rd and 634 row of 186-189, preferred Table II the 3rd and 5 row, 186-189 is capable and/or the 633rd compare with polypeptide expressed activity shown in 634 row and under the same conditions and basic not reduce.In one embodiment, polypeptide of the present invention be comprise as Table II B the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in sequence or by its homologue of forming.

[0189.0.22.22] as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the homologous compound of sequence, or deutero-as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the homologue of sequence also refer to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

Above [0190.0.0.22] and [0191.0.0.22] sees for disclosing of [0190.0.0.22] and [0191.0.0.22] these paragraphs [0190.0.0.0] and [0191.0.0.0]

[0191.1.0.22]: see [0191.1.0.0] for disclosing of this paragraph

[0192.0.0.0] was to [0203.0.0.0] above [0192.0.0.22] to [0203.0.0.22] saw for disclosing of [0192.0.0.22] to [0203.0.0.22] these paragraphs

[0204.0.22.22] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B 186-189 is capable and/or the 633rd and 634 row shown in polypeptide or its segmental nucleic acid molecule, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside quantity increase;

(b) comprise, preferably comprise mature form at least as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B 186-189 is capable and/or the 633rd and 634 row shown in nucleic acid molecule or its segmental nucleic acid molecule, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprise by use as Table III the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in primer or primer nucleic acid molecule that amplification from cDNA library or genomic library is obtained, and in biological or its part, give each fine chemicals, promptly lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably increase of galactolipid and/or cerebroside quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 7th row, 186-189 capable shown in consensus sequence and to give each fine chemicals in biological or its part be lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity;

(k) coded polypeptide aminoacid sequence and to give each fine chemicals in biological or its part be lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity, described peptide coding as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table II B 186-189 is capable and/or the 633rd and 634 row shown in the structural domain of polypeptide; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7,186-189 capable and/or the 633rd and 634 the row shown in nucleic acid molecule or coding (optimized encoding is mature form at least) as Table II the 5th or 7 row, at least the 15nt of nucleic acid molecule of polypeptide shown in capable and/or the 633rd and 634 row of 186-189, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Thereby, the nucleic acid molecule of preferred (a) to (l) by one or more Nucleotide be different from as Table I A or IB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence.In one embodiment, nucleic acid molecule of the present invention not by Table I A or IB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence form.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode as Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in peptide sequence.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide at least one or the different polypeptide of a plurality of amino acid.In another embodiment, Table I A or IB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in nucleic acid molecule do not encode as Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the protein of sequence.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence form.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in protein sequence at least 30% identical and with Table I A or IB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

Above [0205.0.0.22] and [0206.0.0.22] sees for disclosing of [0205.0.0.22] and [0206.0.0.22] these paragraphs [0205.0.0.0] and [0206.0.0.0]

[0207.0.22.22] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is glycolipid, preferably galactose lipid metabolism, semi-lactosi metabolism, fatty acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism, triacylglycerol or lipid, preferably glycerine phosphatide, sphingolipid and/or the biosynthetic gene of galactolipid or its combination.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.0] was to [0226.0.0.0] above [0208.0.0.22] to [0226.0.0.22] saw for disclosing of [0208.0.0.22] to [0226.0.0.22] these paragraphs

[0227.0.22.22] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except the sequence or derivatives thereof that Table I the 5th or 7 is listed as, 186-189 is capable and/or the 633rd and 634 row are mentioned, can advantageously in biology, additionally express and/or other genes that suddenly change.Particularly advantageously, additionally in biological (as plant or microorganism) express that at least one galactolipid is synthetic, other genes of semi-lactosi approach or triacylglycerol or lipid, preferably glycerine fat, sphingolipid, ceramide and/or cerebroside biosynthetic pathway.Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes that increase is synthetic amino acid needed, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0229.0.22.22] sees [0229.0.5.5] for disclosing of this paragraph

[0230.0.0.22] sees [0230.0.0.0] for disclosing of this paragraph

[0231.0.22.22] is in another advantageous embodiment of the inventive method, the employed biology of present method be weakened degraded lipid, preferred glycolipid simultaneously, comprised the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic protein, especially those biologies of the expression rate by reducing corresponding gene.

[0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.22] to [0276.0.0.22] saw for disclosing of [0232.0.0.22] to [0276.0.0.22] these paragraphs

[0277.0.22.22] can separate the compound that comprises glycolipid, preferably galactose fat or semi-lactosi that produces according to the inventive method by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.The fine chemicals that produces according to the inventive method can be as previously mentioned from biological, advantageously from plant with the isolated in form of glycolipid, preferably galactose fat and/or free fatty acids.The compound of producing by this process that comprises glycolipid, preferably galactose fat or semi-lactosi can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing or extract realization with plant part, preferred plant seed.Preferred hexane wherein can separate more than 96% of compound that process of the present invention produces as the solvent in this process.Different with phosphatide, galactolipid is soluble in acetone, utilizes this fact by can easily galactolipid being separated with phosphatide by adsorption chromatography usually.Other organic solvent also is useful as hydrocarbon polymer, chloroform, benzene, ether and alcohol.Afterwards, resulting product is further processed, promptly come unstuck, make with extra care, bleaching and/or deodorizing.

[0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.22] to [0282.0.0.22] saw for disclosing of [0278.0.0.22] to [0282.0.0.22] these paragraphs

[0283.0.22.22] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, particularly anti-as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in proteinic antibody, for example anti-as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the antibody of polypeptide, it can comprise above-mentioned sequence or by its polypeptide of forming by the standard technique utilization, and polypeptide for example of the present invention or its fragment produce.Preferably specificity in conjunction with Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide, more preferably specificity in conjunction with Table II the 5th row, 186-189 is capable and/or the 633rd and 634 row shown in the monoclonal antibody of polypeptide.

[0284.0.0.22] sees [0284.0.0.0] for disclosing of this paragraph

[0285.0.22.22] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in sequence or by as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.22.22] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in capable and/or the 633rd and 634 row of 186-189 or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence or by its polypeptide of forming shown in capable and/or the 633rd and 634 row of 186-189, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, 186-189 is capable and/or the 633rd and 634 row shown in the polypeptide of consensus sequence (each row).

[0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.22] to [0290.0.0.22] saw for disclosing of [0287.0.0.22] to [0290.0.0.22] these paragraphs

[0291.0.22.22] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, 186-189 capable and/or the 633rd and 634 the row shown in sequence, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, 186-189 capable and/or the 633rd and 634 the row shown in sequence.In another embodiment, described polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence form.

[0292.0.0.22] sees [0292.0.0.0] for disclosing of this paragraph

[0293.0.22.22] the present invention relates to give that each fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB, 186-189 capable and/or the 633rd and 634 capable shown in other sequence of sequence phase region.In another embodiment, polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in sequence form.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide not by Table I A or IB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the coded sequence of nucleic acid molecule form.

[0294.0.22.22] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, the polypeptide of activity of proteins shown in capable and/or the 633rd and 634 row of 186-189, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, 186-189 capable and/or the 633rd and 634 the row shown in sequence.

[0295.0.0.0] was to [0297.0.0.0] above [0295.0.0.22] to [0297.0.0.22] saw for disclosing of [0295.0.0.22] to [0297.0.0.22] these paragraphs

The chemical of [00297.1.22.22] non-polypeptide of the present invention be for example do not have Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the active polypeptide of polypeptide.

[0298.0.22.22] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in the abundant homologous aminoacid sequence of aminoacid sequence, give the present invention active ability thereby protein or its part have kept.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 the row shown in the identical aminoacid sequence of sequence.

[0299.0.22.22] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in one of aminoacid sequence sequence homology be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.22.22] is therefore, and be described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 capable shown in sequence different.Therefore, this polypeptide contain with as Table II A or IIB the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the complete amino acid sequence homology of sequence be at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.22] sees [0301.0.0.0] for disclosing of this paragraph

The biologically-active moiety of [0302.0.22.22] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, 186-189 capable and/or the 633rd and 634 the row shown in aminoacid sequence or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.22] sees [0303.0.0.0] for disclosing of this paragraph

[0304.0.22.22] operation nucleic acid molecule of the present invention may cause generation have basically as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.0] was to [0308.0.0.0] above [0305.0.0.22] to [0308.0.0.22] saw for disclosing of [0305.0.0.22] to [0308.0.0.22] these paragraphs

[0306.1.0.22]％

[0309.0.22.22] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, 186-189 capable and/or the 633rd and 634 the row shown in protein be meant polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " non-polypeptide of the present invention " or " other polypeptide " polypeptide of being meant aminoacid sequence in capable and/or the 633rd and 634 row of 186-189 with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, polypeptide shown in capable and/or the 633rd and 634 row of 186-189 is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, proteinic and shown in capable and/or the 633rd and 634 row of 186-189 from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, 186-189 is capable and/or the polypeptide non-of the present invention of the 633rd and 634 row " or " other polypeptide " do not give in biology or its part each fine chemicals and increase.

[0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.22] to [0334.0.0.22] saw for disclosing of [0310.0.0.22] to [0334.0.0.22] these paragraphs

[0335.0.22.22] confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in nucleotide sequence and/or the expression of its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.22] to [0342.0.0.22] saw for disclosing of [0336.0.0.22] to [0342.0.0.22] these paragraphs

[0343.0.22.22] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown between the genetic transcription thing of the nucleotide sequence of one of sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the sequence of one of sequence or the dsRNA that its homologue begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.0] was to [0361.0.0.0] above [0344.0.0.22] to [0361.0.0.22] saw for disclosing of [0344.0.0.22] to [0361.0.0.22] these paragraphs

[0362.0.22.22] therefore, the any nucleic acid that the present invention relates to be used for to be characterized as the present invention's part (for example giving cell or biological or each fine chemicals of its part increases) carry out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide (for example as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide) nucleic acid molecule.Because above-mentioned activity, the fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.In one embodiment, have as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the transgenosis of polypeptide of polypeptide active be meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II the 3rd row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide (for example have as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide of sequence) activity be enhanced.Example and the inventive method are described in above.

[0363.0.0.22] sees [0363.0.0.0] for disclosing of this paragraph

[0364.0.22.22] naturally occurring expression cassette---for example naturally occurring coding such as Table II the 3rd row and/or 5, the 186-189 are capable and/or the 633rd and 634 row shown in the promotor and the combination that is listed as protein coding sequence shown in capable and/or the 633rd and 634 row of 186-189 accordingly as Table I the 3rd and/or 5 of gene of polypeptide of the present invention---become transgene expression cassette.

[0365.0.0.0] was to [0373.0.0.0] above [0365.0.0.22] to [0373.0.0.22] saw for disclosing of [0365.0.0.22] to [0373.0.0.22] these paragraphs

[0374.0.22.22] contain in the methods of the invention institute's synthetic lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic transgenic plant can directly be introduced to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Lipid, the preferred glycolipid that produces by this method, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

Above [0375.0.0.22] and [0376.0.0.22] sees for disclosing of [0375.0.0.22] and [0376.0.0.22] these paragraphs [0375.0.0.0] and [0376.0.0.0]

[0377.0.22.22] therefore the invention still further relates to the method according to this invention, separated the lipid that is produced, preferred glycolipid thus, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside or the fine chemicals that produced.

[0378.0.22.22] can separate by this way and be higher than 50% by weight, favourable is higher than 60%, preferably be higher than 70%, especially preferably be higher than 80%, extremely preferably be higher than 90% the lipid that produces in the method, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.As required, subsequently can be with resultant lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside are further purified, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.22.22] in one embodiment, fine chemicals, particularly lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside are the mixtures that comprises one or more each fine chemicals.In one embodiment, each fine chemicals meaning here be meant lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.In one embodiment, lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside the meaning here are meant the mixture of each fine chemicals.

The lipid that [0380.0.22.22] obtains in the method, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or the suitable parent material of cerebroside as other valuable product.For example they can combinations with one another or are used to produce medicine, food, animal-feed or makeup separately.Liposome and pharmaceutical carrier in the fine chemicals useful as drug preparation of the present invention are called

(the US patent No. 5688528; 5716639,6022561 and 6,068,860).

The galactopyranose base is used to produce oligosaccharides, and they self do not have antigenic action, if but they are when combining with suitable polymer carrier then have antigenic characteristic.For example, " haptens " with sialic acid-gala pyranose base key is described in US 5,296, in 594 and 5,854,218 or 4,686,193.

Be used to reduce the supplement (US 6,126,961) that Salmonellas and other bacterial pathogens are settled in animal intestinal in semi-lactosi or the galactose derivate animal feedstuff compositions.The oligosaccharides that comprises semi-lactosi can be used for preventing mammalian cell to be invaded by pathogenic agent and infect and can be used for overcoming the disease that this type of pathogenic agent causes, and this is described among the US 20050004070.

Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises lipid, preferred glycolipid that separation produces, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside composition or fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is the lipid, the preferred glycolipid that produce of the inventive method, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine.

Above [0381.0.0.22] and [0382.0.0.22] sees for disclosing of [0381.0.0.22] and [0382.0.0.22] these paragraphs [0381.0.0.0] and [0382.0.0.0]

[0383.0.22.22] to contain lipid, preferred glycolipid in order preparing, to comprise the glycolipid of semi-lactosi, the more preferably fine chemicals of galactolipid and/or cerebroside compound, fine chemicals particularly, can use organic compound, the lipid acid that for example contains as precursor compound (for example has C ₁₀--C ₁₆The lipid acid of carbon atom main chain) and/or the lipid of little organic acid (for example acetate, propionic acid or butyric acid) as lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside source.

[0384.0.0.22] sees [0384.0.0.0] for disclosing of this paragraph

The fermented liquid that [0385.0.22.22] obtains with this kind approach, particularly contain with other lipid, fat and/or oils blended lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic fermented liquid, dry matter content accounts for 7.5 to 25% of weight usually.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant) or these methods or partly from fermented liquid, shift out or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.22.22] yet, can also be further purified generation lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.For this reason, the composition that will contain product carries out thin-layer chromatography or carries out Florisil column chromatography (Bouhours J.F., J.Chromatrogr.1979,169,462) on silica-gel plate, and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.Alternatively, the lipid purifying can as

Deng, 1999, Science 284, carry out described in the 2181-2184.

[0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.22] to [0392.0.0.22] saw for disclosing of [0387.0.0.22] to [0392.0.0.22] these paragraphs

[0393.0.22.22] the present invention relates to identify and gives the method that the compound that becomes more meticulous in the cell produces the gene product that increases that it comprises following step in one embodiment:

(a) sample that will comprise candidate gene (be coded in and give the gene product that fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify with nucleic acid molecule of the present invention particularly as Table I the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row, preferred Table I B the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the nucleic acid molecule nucleic acid molecule of under lax stringent condition, hybridizing, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.0] was to [0399.0.0.0] above [0394.0.0.22] to [0399.0.0.22] saw for disclosing of [0394.0.0.22] to [0399.0.0.22] these paragraphs

[00399.1.22.22] it is contemplated that by for example seek to the resistance of blocking-up fine chemicals synthetic medicine and observe this effect whether depend on as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in polypeptide or its homologue active or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, 186-189 is capable and/or the 633rd and 634 row shown in the biology phenotype more much at one of activity of proteins.

[0400.0.0.0] was to [0416.0.0.0] above [0400.0.0.22] to [0416.0.0.22] saw for disclosing of [0400.0.0.22] to [0416.0.0.22] these paragraphs

[0417.0.22.22] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of fatty acid biosynthetic pathway is had resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block fine chemicals synthetic inhibitor in the described biology.The biological example as fine chemicals synthetic inhibitor or weedicide in microorganism or the plant of blocking-up is a Phenylpyridazinones, for example monometflurazone.

[0418.0.0.0] was to [0423.0.0.0] above [0418.0.0.22] to [0423.0.0.22] saw for disclosing of [0418.0.0.22] to [0423.0.0.22] these paragraphs

[0424.0.22.22] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce fine chemicals or produce fine chemicals and one or more other lipids, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside with and composition thereof or with the mixture of other glycolipid.Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing the fine chemicals of biological or its part (as cell).

[0425.0.0.0] was to [0435.0.0.0] above [0425.0.0.22] to [0435.0.0.22] saw for disclosing of [0425.0.0.22] to [0435.0.0.22] these paragraphs

[0436.0.22.22] can change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that [0436.1.22.22] external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces the expection fine chemicals by screening can screen positive catastrophic event.

[0437.0.0.0] to [0453.0.0.0] and [0451.1.0.0] above [0437.0.0.22] to [0453.0.0.22] and [0451.1.0.22] see for disclosing of [0437.0.0.22] to [0453.0.0.22] and [0451.1.0.22] these paragraphs

[0454.0.22.22] embodiment 8: analyzing nucleic acid molecules to lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably influence that produces of galactolipid and/or cerebroside

Above [0455.0.0.22] and [0456.0.0.22] sees for disclosing of [0455.0.0.22] and [0456.0.0.22] these paragraphs [0455.0.5.5] and [0456.0.0.0]

[0457.0.22.22] embodiment 9: by

Deng, 1999, Science 284, the thin-layer chromatography purifying lipid described in the 2181-2184, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside

[0458.0.22.22] embodiment be analyze lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside (abbreviation: FAME, fatty acid methyl ester; GC-MS, gas-liquid chromatograph/mass spectrum; TAG, triacylglycerol; TLC, thin-layer chromatography).By using standard method of analysis GC, GC-MS or TLC can clearly detect the existence of fatty acids products to the biology analysis of recombinating, described analytical procedure Christie and reference wherein are described (1997, Advances on Lipid Methodology, the 4th edition: Christie, Oily Press, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric method], Lipide 33:343-353).The biological total fatty acids that yeast produced that for example is used for the inventive method can be according to the following step analysis: by ultrasonic, the grinding of glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed such as yeast, intestinal bacteria or plant fragmentation.After the fragmentation, must material is centrifugal (1000x g, 10 minutes, 4 ℃) also use 100mM NaHCO ₃, pH 8.0 washings are once to remove residual substratum and lipid acid.In order to prepare fatty acid methyl ester (FAMES), with pellet resuspended in distilled water, in 100 ℃ of heating 10 minutes, cooled on ice is also centrifugal, then in containing the 0.5M sulfuric acid (in methyl alcohol) of 2% Propanal dimethyl acetal, extracted 1 hour in 90 ℃, this causes producing the oil and the fat compound of hydrolysis, and this can obtain transmethylase fat.Then, use 2ml Petroleum ether extraction FAMES twice, use 100mM NaHCO then ₃, pH 8.0 washs once and uses Na ₂SO ₄Dry.Under argon gas stream,, and FAMES heavily is dissolved in the 50 μ l sherwood oils organic solvent evaporation.ZEBRON ZB-Wax capillary column in having Hewlett Packard 6850 gas chromatographs of flame ionization detector (30m, 0,32mm, 0,25 μ m; Phenomenex) go up sample separation.Furnace temperature is carried out time variable control, with 20 ℃/minute speed from 70 ℃ (keeping 1 minute) to 200 ℃, then with 5 ℃/minute speed to 250 ℃ (keeping 5 minutes), at last with 5 ℃/minute speed to 260 ℃.Nitrogen is as carrier gas (4.5ml/ minute, under 70 ℃).The identity of resulting fatty acid methyl ester must be used and can define from the standard that commercial source (being Sigma) obtains.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Then, in 100 ℃ of heating 10 minutes, centrifugation again after the cooled on ice.With cell precipitation in 1M sulfuric acid methanol solution and 2% Propanal dimethyl acetal in 90 ℃ of hydrolysis 1 hour, lipid is by transmethylase.Resulting fatty acid methyl ester (FAME) extracts in sherwood oil.The FAME that is extracted by use capillary column (Chrompack, the WCOT fused silica, CP-Wax-52CB, 25 μ m 0.32mm) carried out gas chromatographic analysis in following 5 minutes in 170 ℃-240 ℃ gradient temperature 20 minutes and 240 ℃.The identity of fatty acid methyl ester must be by relatively confirming with corresponding FAME (being Sigma).Can be suitable chemically derived by the FAME mixture is carried out, for example obtain 4 by GC-MS, 4-Er Jia Yang oxazolin derivs (Christie, 1998) is further identified identity and position of double bond.

Method is described in as Napier and Michaelson, and 2001, Lipids.36 (8): 761-766; Sayanova etc., 2001, Journal of Experimental Botany.52 (360): 1581-1585, Sperling etc., 2001, Arch.Biochem.Biophys.388 (2): 293-298 and Michaelson etc., 1998, FEBS Letters.439 (3): among the 215-218.

[0459.0.22.22] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified lipid, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.Because of the elutriant difference, can advantageously use acetonitrile/water and chloroform/acetonitrile mixture.In order to analyze lipid acid, used ELSD detector (light scattering detector).For the purifying of glycolipid, MPLC, dried post flash chromatography (dry-flash chromatography) or thin-layer chromatography are other favourable chromatographic processes.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.22.22] depends on the fine chemicals that is produced in addition, can also use crystallization and distillation to come purifying.These two kinds of methods are that those skilled in the art are well-known.

[0461.0.22.22] embodiment 10: clone SEQ ID NO:14354 is used for expressing plant

[0462.0.0.0] was to [0466.0.0.0] above [0462.0.0.22] to [0466.0.0.22] saw for disclosing of [0462.0.0.22] to [0466.0.0.22] these paragraphs

[0467.0.22.22] selects following primer sequence for genes of SEQ ID NO:14354:

I) forward primer (SEQ ID NO:14356)

atggcggttg?cgatcaaaaa?gga

Ii) reverse primer (SEQ ID NO:14357)

tcaattgata?aatgtacttt?caatgatg

[0468.0.0.0] was to [0479.0.0.0] above [0468.0.0.22] to [0479.0.0.22] saw for disclosing of [0468.0.0.22] to [0479.0.0.22] these paragraphs

[0480.0.22.22] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:14354

[0482.0.0.0] was to [0513.0.0.0] above [0481.0.0.22] to [0513.0.0.22] saw for disclosing of [0481.0.0.22] to [0513.0.0.22] these paragraphs

[0514.0.22.22] is for the glycolipid by existing in the gas chromatography-mass spectrum methods analyst genetically modified organism, must extract from the material of genetically modified organism, in the presence of methyl alcohol and mineral acid, the extract hydrolysis be obtained corresponding fatty acid methyl ester then and as each monose part of its methyl hexose pyranoside (methylhexopyranoside).

Primary and secondary amido functional group, hydroxyl and free carboxy functional group are finally by carry out TMSization with the N methyl N trimethylsilyl trifluoroacetamide reaction, obtain formerly TMS (TMS) derivative of the formed methyl hexose of hydrolysing step pyranoside (is example with the galactolipid, formation be methylgalactose pyranoside 4TMS).These compounds can be analyzed by gas chromatography-mass spectrum.

Therefore, the increase of the methyl hexose pyranoside content of TMSization is directly related with the increase of glycolipid content in the genetically modified organism.

The different plants of being analyzed the results are shown in following table:

Table 1

??ORF	Metabolite	Analyte	Method	??Min	??Max
						??YER173W	Galactolipid	Methylgalactose pyranoside 4TMS	??GC	??1.20	??1.70
??YLR224W	Galactolipid	Methylgalactose pyranoside 4TMS	??GC	??1.15	??1.46
						??YLR255C	Galactolipid	Methylgalactose pyranoside 4TMS	??GC	??1.16	??1.30
??b2699	Galactolipid	Methylgalactose pyranoside 4TMS	??GC	??1.15	??1.64
						??YHR072W	Galactolipid	Methylgalactose pyranoside 4TMS	??GC	??1.17	??1.35
??b3129	Galactolipid	Methylgalactose pyranoside 4TMS	??GC	??1.16	??1.32

[0515.0.22.22] the 2nd row have shown that metabolite galactolipid and the 3rd row have shown the analyte of being analyzed.The 5th and 6 row have shown the ratio of metabolite/analyte between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 4th row have been pointed out analytical procedure.

[0516.0.0.0] was to [0530.0.0.0] above [0516.0.0.22] to [0530.0.0.22] saw for disclosing of [0516.0.0.22] to [0530.0.0.22] these paragraphs

[0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.22] to [0530.6.0.22] saw for disclosing of [0530.1.0.22] to [0530.6.0.22] these paragraphs

[0531.0.0.0] was to [0552.0.0.0] above [0531.0.0.22] to [0552.0.0.22] saw for disclosing of [0531.0.0.22] to [0552.0.0.22] these paragraphs

[0552.1.0.22]：％

[0552.2.0.22] sees [0552.2.0.0] for disclosing of this paragraph

[0553.0.22.22]

1. produce fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic method, it comprises:

(a) in the activity that improves or produce shown in Table II the 5th or 7 row, 186-189 or 633 and 634 row protein or its function equivalent in non-human being or its one or more parts; With

(b) in allowing described biology, produce fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably cultivate this biology under galactolipid and/or the cerebronic condition.

2. produce fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic method, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, 186-189 or 633 and 634 row, and described nucleic acid molecule gives fat in biology or its part, preferred glycolipid, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside quantity increase;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row, 186-189 or 633 and 634 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity;

E) under stringent hybridization condition, hybridize with nucleic acid molecule (a) to (c) and give fat in biology or its part, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity;

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 186-189 or 633 and 634 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives that fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give fat in biology or its part, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside quantity increase;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 186-189 or 633 and 634 row and give biology or its part in fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprise reclaim free or bonded fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded palmitinic acid of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, 186-189 or 633 and 634 row, and described nucleic acid molecule gives fat in biology or its part, preferred glycolipid, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside quantity increase;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row, 186-189 or 633 and 634 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity;

E) under stringent hybridization condition, hybridize with nucleic acid molecule (a) to (c) and give fat in biology or its part, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside quantity;

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 186-189 or 633 and 634 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives that fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give fat in biology or its part, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside quantity increase;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 186-189 or 633 and 634 row and give biology or its part in fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from sequence shown in Table I A the 5th or 7 row, 186-189 or 633 and 634 row by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, it produces by the method described in claim 13 or by the nucleic acid molecule encoding described in claim 6, this polypeptide is different from sequence shown in Table II A the 5th or 7 row, 186-189 or 633 and 634 row by one or more amino acid thus.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the agonist of screening polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside quantity increase, described method comprises:

(a) will express by the nucleic acid molecule of claim 6 coded and give fat in biology or its part, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably cell, tissue, plant or microorganism and the candidate compound of the polypeptide that increases of galactolipid and/or cerebroside quantity or the sample that comprises multiple compound contact under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps cultivate or keep in the substratum of cell, tissue, plant or microorganism fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside level or expression of polypeptides level; With

(c) fat, the preferred glycolipid by will measuring, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside level or expression of polypeptides level and standard fat, the preferred glycolipid when described candidate compound or the sample that comprises described multiple compound lack, measured, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside or expression of polypeptides level are relatively identified agonist or antagonist; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify give fat in plant or the microorganism, preferred glycolipid, comprise the glycolipid of semi-lactosi, method that more preferably galactolipid and/or cerebroside produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in fat, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably the polypeptide that increases of galactolipid and/or cerebroside quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and it is coded and give fat in biology or its part to be provided at the nucleic acid molecule of described read-out system of permission and claim 6, preferred glycolipid, the glycolipid that comprises semi-lactosi, more preferably respond compound and described polypeptide bonded detectable signal under the condition of the expression of polypeptides of galactolipid and/or cerebroside quantity increase; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify give fat in the cell, preferred glycolipid, comprise the glycolipid of semi-lactosi, method that more preferably galactolipid and/or cerebroside produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, described sample nucleic acid molecule can contain be coded in give fat, preferred glycolipid after the expression, comprise the glycolipid of semi-lactosi, the more preferably candidate gene of the gene product that increases of galactolipid and/or cerebroside;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule import to be fit to is produced fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic host cell;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) analyze fat, preferred glycolipid in the host cell, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside level; With

(f) identify nucleic acid molecule and gene product thereof, compare, give fat in the host cell, preferred glycolipid after it is expressed, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside level improve with wild-type.

20. identify give fat in the cell, preferred glycolipid, comprise the glycolipid of semi-lactosi, method that more preferably galactolipid and/or cerebroside produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain be coded in give fat in biology or its part, preferred glycolipid after the expression, comprise the glycolipid of semi-lactosi, the more preferably candidate gene of the gene product that improves of galactolipid and/or cerebroside quantity or level, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule import to be fit to is produced fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebronic host cell;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) analyze fat, preferred glycolipid in the host living beings, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside level; With

(e) identify nucleic acid molecule and gene product thereof, compare, give fat in the host cell, preferred glycolipid after it is expressed, comprise the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside level improve with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, its be used to identify after expression, give fat, preferred glycolipid, comprise the glycolipid of semi-lactosi, the more preferably nucleic acid molecule that increases of galactolipid and/or cerebroside.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, its be used for identifying can give regulation and control fat, preferred glycolipid at biology, comprise the glycolipid of semi-lactosi, the more preferably compound of galactolipid and/or cerebroside level.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make plant anti-ly to suppress glycolipid, comprises the glycolipid of semi-lactosi, more preferably galactolipid and/or cerebroside synthetic weedicide.

[0554.0.0.22] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.23] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.23] sees top [0001.0.0.0] for disclosing of this paragraph.

[0002.0.23.23] Whitfield's ointment is at whole plants circle ubiquity, and is found in bacterium.It is the important instrumentality of institute's inductive plant disease-resistant substance resistance.

Known a small amount of Whitfield's ointment is present in the plant.Initial Whitfield's ointment is to extract to obtain being used to prepare well-known pain relief medication acetylsalicylic acid from the willow bark.Whitfield's ointment is considered to life-span, the inhibition seed germination that can promote disease resistance, prolong flower and promotes ethene synthetic.

Whitfield's ointment can be synthetic from cinnamate.Before comprised tobacco and other higher plant that the isotropic substance nursing of carrying out in the paddy rice experimental results show that salicylic direct precursor is free phenylformic acid.Phenylformic acid is to shorten the reaction synthetic by the cinnamate chain via the so-called betaoxidation that is similar to the lipid acid β-Yang Hua.By phenylformic acid 2-hydroxylase phenylformic acid is converted into Whitfield's ointment then.That carries out in tobacco recently studies show that the conjugated phenylformic acid, and CoA thioesters or glucose ester might be salicylic precursor more.The Whitfield's ointment that studies show that carries out in mouse ear mustard belongs to more recently can also be synthetic from chorismic acid, and show that a large amount of Whitfield's ointments produce from chorismic acid.

Plant is replied the pathogenic agent attack by activating meticulous defense mechanism.Defensive raction is not only in sites of infection activation, and plant adjacent and even the position activation do not infected of far-end, cause systemic acquired resistance.Plant resistance to environment stress is relevant with the activatory expression of a large amount of defence genes involveds, and the product of defence genes involved has vital role at the restriction pathogenic growth with in propagating.In the past few years, accumulated some evidences and shown that Whitfield's ointment (SA) can be used as the endogenous signal of plant defense reaction.

In most plants, be exposed to mold powder and other pathogenic agent and cause the plant defense approach, recur and help the anti-infectious a series of biological chemistry incidents of plant.Whitfield's ointment is controlled this approach.

When the damage of pathogen resistance and local necrosis was relevant, plant subsequently will be by systematicness " immunity ", will or present the enhanced resistance or presented the symptom that palliates a disease (by Ryals etc., 1996 summarize) to such an extent as to further infect.This kind " systemic acquired resistance " (SAR) expressed relevant (Ward etc., 1992) with the systematicness of one group of defensin gene (the proteinic acid form of relevant PR1-5 of for example curing the disease).To mobilizing the searching of the signal of initiating system resistance to cause Whitfield's ointment (SA) is accredited as most possible candidate from injury region.(possible) pass phloem with very low-level systematicness accumulation before, SA high level around the necrosis damage is synthetic.

[0003.0.23.23] when in the face of fungi or bacterium, most plants are improved its Whitfield's ointment production, and other interaction of molecules of Whitfield's ointment and plant species is then finally opened and produced the anti-infectious proteinic gene of participation.These anti-infective protein also can be closed Whitfield's ointment and be produced, and this is a kind of degenerative phenomenon that is known as.In this kind mode, plant can open and close the pathogenic agent defense pathway as required.

The basic concept that strengthens disease resistance in plants by excessive generation Whitfield's ointment is several years ago by for example Verberne etc., Pharm.World-Sci.; (1995) 17,6 announce.Announced in 2000 after a while that in transgene tobacco expressing intestinal bacteria isochorismate synthase and the different chorismic acid pyruvate salt lyase of fluorescent pseudomonas (Pseudomonas fluorescence) can cause the disease resistance (Verberne that improves, M etc., Nat.Biotechnol.; (2000) 18,7,779-83).These two kinds of enzymes are converted into SA by 2 step processes with chorismic acid.When enzyme target chloroplast(id), to compare with control plant, transgenic plant present SA and the accumulation of SA glucoside improves 500-1,000 times.These plants present the resistance that virus (tobacco mosaic virus (TMV)) and fungi (Oidium lycopersicon) are infected, and are similar to the SAR in the non-transgenic plant.Express the result of two kinds of heterologous genes if this effect is a plastid, so clearly still need alternative or multiple simple method is used for expressing different genes by cytosol and improves Whitfield's ointment production.For indivedual examples or specified plant species, Shi Du Whitfield's ointment increase also may be useful with desired more.

In addition, the transgenic plant (WO2003016551) that salicylic acid binding protein matter can be used for producing the disease resistance with raising have also been described.

Most plants are kept very low-level Whitfield's ointment in its tissue, unless they need resist infection.Yet the too much accumulation of metal can see at the Whitfield's ointment during full time significantly raises its tissue: www.newswise.com/articles/view/510423/.

Nearest result of study is further illustrated in some plant species, and high-level endogenous Whitfield's ointment protective plant is avoided the oxidative stress damage that caused by for example aging or biology or abiotic stress, and (Yang etc., Plant are year December J.2004; 40 (6): 909-19).

[0004.0.23.23] just introduced clinical practice with acetylsalicylic acid before more than 100 years.The medicine of this uniqueness belongs to the compound family that is called salicylate, and wherein the simplest is Whitfield's ointment, and Whitfield's ointment is the major metabolite of acetylsalicylic acid.Whitfield's ointment is given the acetylsalicylic acid anti-inflammatory effect, and plants the crowd who takes acetylsalicylic acid and to observe Whitfield's ointment and can cause that colorectal carcinoma is dangerous and reduce.Yet, Whitfield's ointment and other salicylate is natural is present in fruit and the plant, and the diet that is rich in these materials is believed and can be reduced colorectal carcinoma danger.Vegetarian's serum Whitfield's ointment concentration is higher than the nonvegetarian, and the vegetarian with take between the concentration among the people of low dosage acetylsalicylic acid overlapped.The preventing cancer effect that should propose acetylsalicylic acid is owing to its major metabolite, Whitfield's ointment, and the diet salicylate may have identical effect.Natural salicylate also has other the generally acknowledged benefit that helps health diet.

Salicylic oh group of [0005.0.23.23] T and acetic acid reaction form acetic ester, acyl group Whitfield's ointment (seeing acetylsalicylic acid).Form several useful esters by carboxyl and ethanol synthesis.Methyl ester, cresotinic acid acid esters (so because its fragrance that produces teaberry is also referred to as oil of wintergreen) forms with methyl alcohol; It is used for food-flavoring comps and liniment.Phenylester, phenyl salicylic acid esters or salol form with phenol; It is used for medical science as sanitas and febrifugee.The hydrolysis in the intestines of alkalescence rather than in the tart stomach of this kind ester discharges free salicylic acid.Be used to shine the menthyl ester of black wash, the menthyl salicylate forms with menthol.

Whitfield's ointment has the bacterium of causing, fungicidal and cutin solvency action.

[0006.0.23.23] Whitfield's ointment is as food preservatives and be used as antiseptic-germicide in toothpaste.It is the furfur agent of ointment, emulsifiable paste, gelifying agent and shampoo that is used for reducing the fouling of psoriatic patient skin or scalp.Because Whitfield's ointment can cause more easily that skin cells comes off, prevent their obstruction pores, it is the activeconstituents in many treatment acnes ground skin products.

[0007.0.23.23] Whitfield's ointment belongs to the pharmaceutical that is known as keratolytic agent.Whitfield's ointment works by the degraded Keratin sulfate, and Keratin sulfate is the protein that forms the skin texture part.This causes skin cells to come off from infected position.In the treatment of boss, scleroma and wart, salicylic effect is to remove infected skin in for some time.If success, Sheng Chang new skin will be healthy below.

A kind of method that [0008.0.23.23] improves biosynthesizing throughput is to use recombinant DNA technology.Therefore, people are desirably in and produce Whitfield's ointment and/or salicylate in the plant.The inhuman production of this type allows the most suitable and the most effective quality, quantity and the screening of producing biology of control.The latter is particularly important for commercial production economics, and also is favourable for the human consumer therefore.In addition, people are desirably in and produce Whitfield's ointment in the plant, so that improve plant production power and to the resistance of biological and abiotic stress as mentioned above.

More already used year of recombinant DNA technology method is with by increasing different biosynthesis genes and study the production that its influence to fine chemicals production improves fine chemicals in microorganism and the plant.For example, it is reported that the xenthophylls astaxanthin can result from the nectary of rotaring gene tobacco plant.Those transgenic plant are to prepare by the carrier transformation of tobacco plant that agrobacterium tumefaciens mediates, and wherein carrier comprises the ketolase encoding gene (called after crtO) from H.pluvialis, and the Pds gene of tomato is as the promotor and the leader sequence of encoding.Those results show that about 75% carotenoid that is found in the conversion plant flowers contains ketone group.

[0009.0.23.23] if can obtain producing a large amount of salicylic algae, plant or other microorganism, should be favourable therefore.The protokaryon or the eukaryotic microorganisms that relate to this type of conversion in some embodiments that the present invention is hereinafter discussed.

If it also should be favourable can obtaining root, leaf, stem, fruit or spend a large amount of salicylic plants of middle generation.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

In addition, also should be favourable if can obtain producing in the seed a large amount of plants of lipids always.Relate to this type of plant transformed in some embodiments that the present invention is hereinafter discussed.

The quality that [0010.0.23.23] therefore improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the Whitfield's ointment that for example is present in as mentioned above in plant and some microorganisms is limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is specific Whitfield's ointment spectrum in the balanced diet as far as possible, because excessive Whitfield's ointment has negative effect on the specific concentrations.Further improving the quality only may be by adding other Whitfield's ointment, and these compositions all are limited.

[0011.0.23.23] is necessary with balance mode Whitfield's ointment and/or salicylate to be added into suitable biology in order to guarantee the high quality of food and animal-feed.

[0012.0.23.23] therefore still is starved of one or more codings and participates in the biosynthetic enzyme of Whitfield's ointment or other proteinic suitable gene, and make and might not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but forever need to improve the method that obtains the highest intrinsic energy Whitfield's ointment and/or salicylate; On the other hand, reduce the byproduct that produces in the production process as far as possible.

In addition, still be starved of one or more codings and participate in the biosynthetic enzyme of total lipid or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but need forever to improve the method that obtains the total lipid of the highest intrinsic energy, on the other hand, reduce the byproduct that produces in the production process as far as possible.

[0013.0.0.23] sees [0013.0.0.0] for disclosing of this paragraph

[0014.0.23.23] therefore in the first embodiment, the present invention relates to produce the method for fine chemicals, and fine chemicals is Whitfield's ointment or salicylate thus.In addition, as used herein term " fine chemicals " also refers to comprise the fine chemicals of Whitfield's ointment and/or salicylate.

In one embodiment, term " fine chemicals " meaning is meant Whitfield's ointment.In whole specification sheets, term " fine chemicals " meaning is meant Whitfield's ointment, its salt, ester, thioesters or free form or is bonded to the form of other compound (for example sugar).

[0015.0.23.23] especially, the technician knows, anionic compound such as acid are present in the aqueous solution with the balance between sour and its salt according to the pK value of pH value in each chamber in cell or the organism and acid.Depend on the intensity (pK) and the pH of acid, dominant is salt or free acid.Therefore, term " fine chemicals ", term " each fine chemicals ", term " acid " or mention in the title used during with anionic compound relevant with the anionic form and the neutral condition (relevant) of compound with the condition of the aqueous solution.

[0016.0.23.23] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0161, b2664, b2796, b3116 or YLR089C protein; With

(b) producing fine chemicals in allowing described biology is to make biological growth under the salicylic condition.Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has activity of proteins shown in Table II the 3rd row, 275-277 and/or 635 or 636 row, perhaps have by Table I the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce under the condition that fine chemicals is Whitfield's ointment and/or salicylate and make biological growth.

[0016.1.23.23] therefore, term " fine chemicals " meaning is meant and Table I the 5th or 7 row, 275-277 and/or relevant " Whitfield's ointment " or its homologue of the 635 or 636 listed full sequences of row.

Above [0017.0.0.23] and [0018.0.0.23] sees for disclosing of [0017.0.0.23] and [0018.0.0.23] these paragraphs [0017.0.0.0] and [0018.0.0.0]

The method that [0019.0.23.23] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in 275-277 and/or 635 or 636 row or by as Table I the 5th or 7 be listed as, the activity of proteins of the coded protein active of nucleic acid molecule is carried out above-mentioned modification shown in 275-277 and/or 635 or 636 row.

[0020.0.23.23] is surprisingly found out that, Whitfield's ointment (with regard to described protein and homologue and coding nucleic acid molecule (particularly shown in Table II the 3rd row, 275-277 and/or 635 or 636 row), being " fine chemicals " or " each fine chemicals ") content that e. coli k12 protein b0161, b2796, b3116 or b2664 or yeast saccharomyces cerevisiae protein YLR089C give the plant that transforms at transgene expression in the Arabidopis thaliana increases.

Be surprisingly found out that yeast saccharomyces cerevisiae protein YLR089C and/or e. coli k12 protein b0161, b2796, b3116 and/or b2664 transgene expression in Arabidopis thaliana shown in Table II the 3rd or 5 row, the 275th, 276,635 and/or 636 row are given the institute's Whitfield's ointment of conversion plant and/or the increase of salicylate content shown in Table II the 3rd or 5 row, the 277th row.

Therefore, in one embodiment, described protein or its homologue (shown in Table II the 7th row, 275-277 and/or 635 or 636 row) are used to produce Whitfield's ointment and/or salicylate.

[0021.0.0.23] sees [0021.0.0.0] for disclosing of this paragraph

The sequence of [0022.0.23.23] e. coli k12 b0161 has been published in Blattner, F.R. etc., Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be the protein with serine protease.Therefore, in one embodiment, the inventive method comprises as shown here from the protein b0161 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases Whitfield's ointment, the preferred salicylic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b0161 is enhanced.

The sequence of e. coli k12 b2664 has been published in Blattner, F.R. etc., Science 277 (5331), 1453-1474 (1997), and its activity to be defined as be to have the active protein of transcription repressor (GntR family).Therefore, in one embodiment, the inventive method comprises as shown here from the protein b2664 of e. coli k12 or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases Whitfield's ointment, the preferred salicylic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b2664 is enhanced.

The sequence of e. coli k12 b2796 (accession number NP_417276) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition Serine translocator (HAAAP family).Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having Threonine-active gene product of Serine permease superfamily, preferably having Serine translocator (HAAAP family) the active protein of supposition or a purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, i.e. Whitfield's ointment, the preferred salicylic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b2796 is enhanced.

The sequence of e. coli k12 b3116 (accession number NP_417586) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the L-Threonine/L-Serine permease (HAAAP family) of anaerobic induction.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillaryly having Threonine-active gene product of Serine permease superfamily, preferably having L-Threonine/L-Serine permease (HAAAP family) the active protein of anaerobic induction or a purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, i.e. Whitfield's ointment, the preferred salicylic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of protein b3116 is enhanced.

The sequence of yeast saccharomyces cerevisiae YLR089C has been published in Johnston, M. etc., Nature 387 (6632 supplementary issue), 87-90 (1997), and its activity to be defined as be that protein has alanine aminotransferase (gpt) activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein YLR089C of yeast saccharomyces cerevisiae or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, particularly increases Whitfield's ointment, the preferred salicylic quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of protein YLR089C is enhanced.

The homologous compound (=homologue) of [0023.0.23.23] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, the homologue of polypeptide is to have same or similar active homologue shown in Table II the 3rd row, 275-277 and/or 635 or 636 row.Particularly, each fine chemicals in the biology is given in active raising, preferred salicylic acid content increases.

The homologue of polypeptide shown in Table II the 3rd row, 275-277 and/or 635 or 636 row can be to have the salicylic acid content of increasing and/or an active polypeptide of quantity by nucleic acid molecule encoding shown in Table I the 7th row, 275-277 and/or 635 or 636 row.

The homologue of polypeptide shown in [0023.1.23.23] Table II the 3rd row, 275-277 and/or 635 or 636 row can be by the polypeptide of nucleic acid molecule encoding shown in Table I the 7th row, 275-277 and/or 635 or 636 row or can be polypeptide shown in Table II the 7th row, 275-277 and/or 635 or 636 row.

[0024.0.0.23] sees [0024.0.0.0] for disclosing of this paragraph

[0025.0.23.23] is according to the present invention, the Whitfield's ointment level increases in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example activity of proteins shown in Table II the 3rd row, 275-277 and/or 635 or 636 row.In preferred embodiments, protein or polypeptide have proteinic above-mentioned extra activity shown in Table II the 3rd row, 275-277 and/or 635 or 636 row.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, any one proteinic biology or the enzyme activity shown in 275-277 and/or 635 or 636 row, if promptly with Table II the 3rd row, the 275th, 276, shown in e. coli k12 protein shown in 635 and/or 636 row or the 277th row in the yeast saccharomyces cerevisiae protein any one compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as each fine chemicals of increase in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

Above [0025.1.0.23] and [0025.2.0.23] sees for disclosing of [0025.1.0.23] and [0025.2.0.23] these paragraphs [0025.1.0.0] and [0025.2.0.0]

[0026.0.0.0] was to [0033.0.0.0] above [0026.0.0.23] to [0033.0.0.23] saw for disclosing of [0026.0.0.23] to [0033.0.0.23] these paragraphs

[0034.0.23.23] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, its have protein shown in Table II the 3rd row, 275-277 and/or 635 or 636 row or by the protein of nucleic acid molecule encoding shown in Table I the 5th row, 275-277 and/or 635 or 636 row or its homologue (homologue shown in Table I the 7th row, 275-277 and/or 635 or 636 row) active protein expression level or active aspect difference, and its difference aspect biological chemistry or genetics reason.Therefore it shows each fine chemicals quantity that increases.

[0035.0.0.0] was to [0044.0.0.0] above [0035.0.0.23] to [0044.0.0.23] saw for disclosing of [0035.0.0.23] to [0044.0.0.23] these paragraphs

[0045.0.23.23] in one embodiment, under the situation that the activity of e. coli k12 protein b0161 or its homologue (for example shown in Table II the 5th or 7 row, the 275th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Whitfield's ointment be increased in 67% and 475% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2664 or its homologue (for example shown in Table II the 5th or 7 row, the 276th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Whitfield's ointment be increased in 56% and 425% or more between.

In one embodiment, under the situation that the activity of yeast saccharomyces cerevisiae protein YLR089C or its homologue (for example shown in Table II the 5th or 7 row, the 277th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Whitfield's ointment be increased in 45% and 153% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b2796 or its homologue (for example shown in Table II the 5th or 7 row, the 635th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Whitfield's ointment be increased in 42% and 248% or more between.

In one embodiment, under the situation that the activity of e. coli k12 protein b3116 or its homologue (for example shown in Table II the 5th or 7 row, the 636th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred Whitfield's ointment be increased in 41% and 130% or more between.

[0046.0.0.23]％

Above [0047.0.0.23] and [0048.0.0.23] sees for disclosing of [0047.0.0.23] and [0048.0.0.23] these paragraphs [0047.0.0.0] and [0048.0.0.0]

[0049.0.23.23] has to give and improves each fine chemicals Whitfield's ointment quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise the NO:33804 as SEQ ID, 33805,33806,33807,33808,33809 or 33896,33897,33898 or 34219,34220,34221,34222,34223,34224,34225,34226,34227 or 99065,99066,99067,99170,99171,99172,99173,99174,99175,99176, shown in 99177 or Table IV the 7th row, the sequence of consensus sequence shown in 275-277 and/or 635 or 636 row or as Table II the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in 275-277 and/or 635 or 636 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in 275-277 and/or 635 or 636 row) sequence of coded polypeptide, and have the activity that the Whitfield's ointment level of giving described in the literary composition increases.

[0050.0.23.23] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt (for example salicylic sylvite or sodium salt) or their ester (such as but not limited to methyl ester, phenylester or menthol ester).

[0051.0.23.23] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with Whitfield's ointment), for example comprise salicylic composition.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of Whitfield's ointment and salicylate.

[0052.0.0.23] sees [0052.0.0.0] for disclosing of this paragraph

[0053.0.23.23] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, protein or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row) expression that described protein is given nucleic acid molecule encoding of the present invention increase, and have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row) and are expressed and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by coded protein of nucleic acid molecule of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row), perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) produce or improve the endogenous transcription factor that mediating protein expresses or the expression of manual transcription factor, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by nucleic acid molecule of the present invention coded protein or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row);

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by nucleic acid molecule of the present invention coded protein or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row);

(f) express the transgenosis of coded protein, described protein give have each fine chemicals of the raising described in the literary composition active, express and improve by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row);

(g) copy number of raising gene, described gene is given nucleic acid molecule and express to be improved, described nucleic acid molecule encoding have each fine chemicals of the raising described in the literary composition active, by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row);

(h), for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element by adding positive Expression element or removing the expression that negative Expression element improves the native gene of code book invention polypeptide (for example having protein or the active polypeptide of its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) shown in Table II the 3rd row, 275-277 and/or 635 or 636 row).Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.23.23] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improve coded protein or have shown in Table II the 3rd or 5 row, 275-277 and/or 635 or 636 row protein or its homologue (for example polypeptide shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) active expression of polypeptides or activity after give each fine chemicals and increase.

[0055.0.0.0] was to [0067.0.0.0] above [0055.0.0.23] to [0067.0.0.23] saw for disclosing of [0055.0.0.23] to [0067.0.0.23] these paragraphs

[0068.0.23.23] be not can cause the mode of disadvantageous effect to introduce sudden change to salicylic generation.

[0069.0.0.23] sees [0069.0.0.0] for disclosing of this paragraph

[0070.0.23.23] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, the composition of for example favourable Whitfield's ointment or their biological chemistry derivative, for example contain the composition of high level (from the physiology of nutrition angle) Whitfield's ointment or their derivative, include but not limited to salicylate.

[0071.0.0.23] sees [0071.0.0.0] for disclosing of this paragraph

[0072.0.0.23]％

[0073.0.23.23] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of polypeptide of the present invention or its homologue (for example shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if (randomly separating) free and/or each fine chemicals of bonded by biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis is reclaimed in expectation.

[0074.0.23.23] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded.

[0075.0.0.0] was to [0077.0.0.0] above [0075.0.0.23] to [0077.0.0.23] saw for disclosing of [0075.0.0.23] to [0077.0.0.23] these paragraphs

[0078.0.23.23] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes.Method well known to those skilled in the art be can use and purified fermentation broth, tunning, plant or plant product come.For these different methods of setting up gradually, product is Whitfield's ointment or salicylate or contains Whitfield's ointment and the composition of salicylate, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.0] was to [0084.0.0.0] above [0079.0.0.23] to [0084.0.0.23] saw for disclosing of [0079.0.0.23] to [0084.0.0.23] these paragraphs

[0085.0.23.23] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) nucleotide sequence or derivatives thereof shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, perhaps

(b) genetic regulatory element that effectively is connected with nucleotide sequence or derivatives thereof shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, promotor for example, perhaps (c) (a) with (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

Above [0086.0.0.23] and [0087.0.0.23] sees for disclosing of [0086.0.0.23] and [0087.0.0.23] these paragraphs [0086.0.0.0] and [0087.0.0.0]

[0088.0.23.23] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Stronger and output is enhanced to the resistance of biological and abiotic stress because the nutritive value that for example is used for the plant of raise poultry depends on above-mentioned fine chemicals or plant, so this is very important for the plant breeder.

[0088.1.0.23] sees [0088.1.0.0] for disclosing of this paragraph

[0089.0.0.0] was to [0094.0.0.0] above [0089.0.0.23] to [0094.0.0.23] saw for disclosing of [0089.0.0.23] to [0094.0.0.23] these paragraphs

[0095.0.23.23] advantageously increases Whitfield's ointment and/or salicylate pond to separate a large amount of pure basically fine chemicals and/or to obtain the resistance of the raising of biological and abiotic stress and obtain high yield by method of the present invention in genetically modified organism.

[0096.0.23.23] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.

[0097.0.0.23] sees [0097.0.0.0] for disclosing of this paragraph

[0098.0.23.23] in preferred embodiments, each fine chemicals be produced according to the present invention and, if the expectation, separated.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.23.23].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition that comprises each fine chemicals.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.23.23] comprises the transgenic plant that the method according to this invention synthetic comprises fine chemicals such as Whitfield's ointment and/or salicylate and can advantageously directly put on market, and do not need institute's synthetic fine chemicals is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet each fine chemicals that the method according to this invention produces can also be as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free fine chemicals from biology, advantageously separate from plant.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because Whitfield's ointment and/or salicylate are positioned at cell or extracellular in the microorganism, must relate to separation to their recovery to biomass or supernatant.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.

[0101.0.23.23] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.23.23] for example, Whitfield's ointment and/or salicylate can separate and MS (mass spectrum) detection method is advantageously analyzed by HPLC, LC or GC.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect Whitfield's ointment and/or containing the existence of salicylic product to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

[0103.0.23.23] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide has the sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) the preferred nucleic acid molecule that comprises the mature form nucleic acid molecule at least, described mature form nucleic acid molecule have the sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer of sequence shown in Table III the 7th row, 275-277 and/or 635 or 636 row obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise consensus sequence with sequence shown in Table IV the 7th row, 275-277 and/or 635 or 636 row and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding Table II the 5th or 7 row, 275-277 and/or 635 or 636 row; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.23.23] in one embodiment, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method is not made up of sequence shown in Table I A the 5th or 7 row, 275-277 and/or 635 or 636 row.In one embodiment, the identity of sequence shown in the inventive method nucleic acid molecule used therefor and Table I A the 5th or 7 row, 275-277 and/or 635 or 636 row is less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II A the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in polypeptide of sequence.

[00103.2.23.23] in one embodiment, nucleic acid molecule of the present invention and Table I B the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method is not made up of sequence shown in Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row.In one embodiment, the identity of sequence shown in the inventive method nucleic acid molecule used therefor and Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row is less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIB the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in polypeptide of sequence.

[0104.0.23.23] in one embodiment, the sequence difference is one or more Nucleotide shown in nucleic acid molecule of the present invention and Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I A the 5th or 7 row, 275-277 and/or 635 or 636 row.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method is not made up of sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I A the 5th or 7 row, 275-277 and/or 635 or 636 row.In one embodiment, the identity of sequence shown in nucleic acid molecule of the present invention and Table I the 5th or 7 row, 275-277 and/or 635 or 636 row is less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in polypeptide of sequence, preferably nucleic acid molecule not coding schedule IIA the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in polypeptide of sequence.

[0105.0.0.0] was to [0107.0.0.0] above [0105.0.0.23] to [0107.0.0.23] saw for disclosing of [0105.0.0.23] to [0107.0.0.23] these paragraphs

Advantageously improved in [0108.0.23.23] method of the present invention and had Table I the 5th or 7 row, the nucleic acid molecule of sequence shown in 275-277 and/or 635 or 636 row, from Table II the 5th or 7 row, aminoacid sequence shown in 275-277 and/or 635 or 636 row is derived or is listed as from containing Table IV the 7th, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 275-277 and/or 635 or 636 row, perhaps its coding has as Table II the 3rd, 5 or 7 row, the enzymic activity of polypeptide or bioactive polypeptide shown in 275-277 and/or 635 or 636 row or for example to give each fine chemicals behind its expression or active the increasing be derivative or the homologue that Whitfield's ointment and/or salicylate increase.

[0109.0.23.23] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make each fine chemicals, particularly Whitfield's ointment and/or salicylate that the inventive method produced be optimized synthetic.

The nucleic acid molecule that [0110.0.0.23] helps the inventive method and coding has the polypeptide that is used for the inventive method or the polypeptide that is used for process of the present invention (for example protein shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row or by coded protein or its homologue of nucleic acid molecule shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) active polypeptide can be determined from generally open database.

[0111.0.0.23] sees [0111.0.0.0] for disclosing of this paragraph

The nucleic acid molecule that uses in [0112.0.23.23] the inventive method is the isolated nucleic acid sequences form, its coding has the polypeptide of polypeptide active shown in Table II the 3rd row, 275-277 and/or 635 or 636 row or has the polypeptide of peptide sequence shown in Table II the 5th and 7 row, 275-277 and/or 635 or 636 row, and gives Whitfield's ointment and/or the increase of salicylate level.

Above [0113.0.0.23] to [0120.0.0.23] sees for disclosing of [0113.0.0.23] to [0120.0.0.23] these paragraphs [0113.0.0.0] and [0120.0.0.0]

[0120.1.0.23]：％

[0121.0.23.23] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, peptide sequence or its functional homologue as herein described have the difference of one or more amino acid moleculars shown in 275-277 and/or 635 or 636 row, described artificial sequence is preferably given aforementioned activity, promptly improving Table II the 5th or 7 row, giving the Whitfield's ointment level after the activity of peptide sequence shown in 275-277 and/or 635 or 636 row increases, and is improving Table II the 5th or 7 row, giving the Whitfield's ointment level after the activity of peptide sequence shown in 275-277 and/or 635 or 636 row increases.

Above [0122.0.0.23] to [0127.0.0.23] sees for disclosing of [0122.0.0.23] to [0127.0.0.23] these paragraphs [0122.0.0.0] and [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.23.23] polymerase chain reaction (PCR) amplification is used (for example the primer shown in Table III the 7th row, 275-277 and/or 635 or 636 row to) can be based on sequence shown in this paper, for example sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row or produce from sequence deutero-sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row.

[0129.0.23.23] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Consensus sequence is compared since then shown in Table IV the 7th row, 275-277 and/or 635 or 636 row.

[0130.0.23.23] can use then degenerated primer by pcr amplification have aforementioned activity (as improve to express or activity after give Whitfield's ointment and/or salicylate increases) the new protein fragment or comprise as described in segmental protein.

[0131.0.0.0] was to [0138.0.0.0] above [0131.0.0.23] to [0138.0.0.23] saw for disclosing of [0131.0.0.23] to [0138.0.0.23] these paragraphs

[0139.0.23.23] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with sequence hybridization shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row, and the coding expression has each fine chemicals, the i.e. active peptide of Whitfield's ointment and/or salicylate of increasing.

[0140.0.0.0] was to [0146.0.0.0] above [0140.0.0.23] to [0146.0.0.23] saw for disclosing of [0140.0.0.23] to [0146.0.0.23] these paragraphs

[0147.0.23.23] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.Be meant these molecules with one of nucleotide sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row complementary nucleic acid molecule: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.23.23] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, nucleotide sequence or its portion homologous are at least about 30% shown in 275-277 and/or 635 or 636 row, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly having after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue increases Whitfield's ointment and/or salicylic activity.

[0149.0.23.23] nucleic acid molecule of the present invention comprises nucleotide sequence, one of nucleotide sequence shown in described sequence and Table I the 5th or 7 row, 275-277 and/or 635 or 636 row or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity (increasing as giving Whitfield's ointment and/or Whitfield's ointment) and the protein of protein active shown in Table II the 5th row, 275-277 and/or 635 or 636 row randomly.

[00149.1.23.23] randomly, in one embodiment, with Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, the nucleotide sequence of one of nucleotide sequence hybridization shown in 275-277 and/or 635 or 636 row has another or various active, described activity is for being listed as Table II the 3rd, protein shown in 275-277 and/or 635 or 636 row is known activity or is used for these protein of note, preferably for being listed as Table II B the 5th or 7, protein shown in 275-277 and/or 635 or 636 row is known activity or is used for these protein of note.

[00149.1.22.22] randomly, have another or various active with the nucleotide sequence of one of nucleotide sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row hybridization, described activity for as Table II the 3rd row, 275-277 is capable and/or the 635th or 636 row, preferred Table II B the 5th or 7 row, 275-277 and/or 635 or 636 row shown in protein be known activity or be used for these protein of note.

[0150.0.23.23] in addition, nucleic acid molecule of the present invention can only contain the part of the coding region of one of nucleotide sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give Whitfield's ointment and/or salicylate increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, described nucleotides sequence column region under stringent condition with the antisense sequences of one of sequence shown in sense strand, Table I the 5th or 7 row, 275-277 and/or 635 or 636 row of one of sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row or its natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotides hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.To produce fragment or its gene product of polynucleotide sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row with the right PCR of primer shown in Table III the 7th row, 275-277 and/or 635 or 636 row.Preferably Table I B the 5th or 7 row, 275-277 and/or 635 or 636 go.

[0151.0.0.23]: see [0151.0.0.0] for disclosing of this paragraph

[0152.0.23.23] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and the abundant homology of aminoacid sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or the activity of raising Whitfield's ointment level as be shown in the examples.

[0153.0.23.23] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, the amino-acid residue that described aminoacid sequence comprises with the aminoacid sequence minimal number is identical or of equal value shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, protein or its part have the activity of polypeptide shown in Table II the 3rd row for example as herein described, 275-277 and/or 635 or 636 row shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row.

[0154.0.23.23] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein is at least about 30%, 35%, 45% or 50% with complete amino acid sequence homology shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about 95%, 96%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

Above [0155.0.0.23] and [0156.0.0.23] sees for disclosing of [0155.0.0.23] and [0156.0.0.23] these paragraphs [0155.0.0.0] and [0156.0.0.0]

[0157.0.23.23] the present invention relate in addition owing to the genetic code degeneracy be different from one of nucleotide sequence (with its part) shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row and thereby code book invention polypeptide, the polypeptide that particularly has aforementioned activity (for example give in the biology each fine chemicals increase) for example comprises shown in Table IV the 7th row, 275-277 and/or 635 or 636 row polypeptide of sequence or the nucleic acid molecule of polypeptide or its function homologue shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, described protein comprise or have in another embodiment shown in Table IV the 7th row, 275-277 and/or 635 or 636 row consensus sequence or shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row the aminoacid sequence of polypeptide or its function homologue.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise shown in Table IV the 7th row, 275-277 and/or 635 or 636 row consensus sequence or shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row the basic homology of aminoacid sequence of polypeptide or its function homologue.Yet in a preferred embodiment, nucleic acid molecule of the present invention is not made up of sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I A the 5th or 7 row, 275-277 and/or 635 or 636 row.Preferably, nucleic acid molecule of the present invention is the functional homologous compound of nucleic acid molecule shown in Table I B the 7th row, 275-277 and/or 635 or 636 row or identical with it.

[0158.0.0.0] was to [0160.0.0.0] above [0158.0.0.23] to [0160.0.0.23] saw for disclosing of [0158.0.0.23] to [0160.0.0.23] these paragraphs

[0161.0.23.23] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (nucleic acid molecule that for example comprises sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.23] sees [0162.0.0.0] for disclosing of this paragraph

[0163.0.23.23] preferably, under stringent condition with the nucleic acid molecule of the present invention of sequence hybridization shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals quantity increase in biology or its part (for example tissue, cell or cell chamber) after using activity of proteins).

[0164.0.0.23] sees [0164.0.0.0] for disclosing of this paragraph

[0165.0.23.23] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence (for example sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row) of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method.

Above [0166.0.0.23] and [0167.0.0.23] sees for disclosing of [0166.0.0.23] and [0167.0.0.23] these paragraphs [0166.0.0.0] and [0167.0.0.0]

[0168.0.23.23] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are different with the contained sequence of sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with aminoacid sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, sequence shown in 275-277 and/or 635 or 636 row is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, one of sequence shown in 275-277 and/or 635 or 636 row is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, sequence shown in 275-277 and/or 635 or 636 row is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, sequence shown in 275-277 and/or 635 or 636 row is at least about 96%, 97%, 98% or 99% is identical.

Therefore, the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), described polypeptide contains the variation to the nonessential amino-acid residue of described activity.The contained sequence of these amino acid sequence of polypeptide and sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table II B the 7th row, 275-277 and/or 635 or 636 row is different, but has kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with aminoacid sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table II B the 7th row, 275-277 and/or 635 or 636 row at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, 275-277 and/or 635 or 636 row, preferred Table II B the 7th row, sequence shown in 275-277 and/or 635 or 636 row is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, 275-277 and/or 635 or 636 row, preferred Table II B the 7th row, one of sequence shown in 275-277 and/or 635 or 636 row is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, 275-277 and/or 635 or 636 row, preferred Table II B the 7th row, sequence shown in 275-277 and/or 635 or 636 row is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, 275-277 and/or 635 or 636 row, preferred Table II B the 7th row, sequence shown in 275-277 and/or 635 or 636 row is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.0] was to [0172.0.0.0] above [0169.0.0.23] to [0172.0.0.23] saw for disclosing of [0169.0.0.23] to [0172.0.0.23] these paragraphs

[0173.0.23.23] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:33502 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:33502 sequence before use.

[0174.0.0.23]: see [0174.0.0.0] for disclosing of this paragraph

[0175.0.23.23] for example, the sequence that has 80% homology at protein level and SEQ ID NO:33503 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:33503 sequence.

[0176.0.23.23] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, the functional equivalent that one of polypeptide obtains shown in 275-277 and/or 635 or 636 row be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in 275-277 and/or 635 or 636 row, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, polypeptide shown in 275-277 and/or 635 or 636 row has essentially identical character and discerns.

[0177.0.23.23] is by replacing, insert or lack from being listed as Table I the 5th or 7 according to of the present invention, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, the functional equivalent that obtains of nucleotide sequence shown in 275-277 and/or 635 or 636 row be listed as according to of the present invention as Table II the 5th or 7, one of polypeptide has at least 30% shown in 275-277 and/or 635 or 636 row, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II the 5th or 7 be listed as, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, the polypeptide of the essentially identical character of polypeptide shown in 275-277 and/or 635 or 636 row.

[0178.0.0.23] sees [0178.0.0.0] for disclosing of this paragraph

[0179.0.23.23] can (particularly be listed as Table I the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, shown in 275-277 and/or 635 or 636 row) the middle replacement of introducing one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, the nucleic acid molecule of the homologous compound of protein sequence shown in 275-277 and/or 635 or 636 row.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row, introducing sudden change in the encoding sequence.

[0180.0.0.0] was to [0183.0.0.0] above [0180.0.0.23] to [0183.0.0.23] saw for disclosing of [0180.0.0.23] to [0183.0.0.23] these paragraphs

[0184.0.23.23] is employed to have as Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, the nucleotide sequence homologous compound of sequence shown in 275-277 and/or 635 or 636 row, perhaps come Table II the 5th or 7 row freely, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, the homologous compound of nucleotide sequence of sequence shown in 275-277 and/or 635 or 636 row also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.23.23] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more sequences shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row.In one embodiment, the preferred nucleic acid molecule comprises other Nucleotide the least possible, that do not show in the arbitrary sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method is identical with sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row.

The employed one or more nucleic acid molecule encodings of [0186.0.23.23] also preferred the inventive method comprise polypeptide of sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, 275-277 and/or 635 or 636 row.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method is identical with sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, 275-277 and/or 635 or 636 row.

[0187.0.23.23] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, 275-277 and/or 635 or 636 row polypeptide of sequence and contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, employed nucleic acid molecule is identical with the encoding sequence of coding sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, 275-277 and/or 635 or 636 row in the method.

[0188.0.23.23] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active comparing with polypeptide expressed activity shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row and under the same conditions do not reduced substantially.

[0189.0.23.23] be the homologous compound of sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, or the homologue of the sequence of deutero-shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

Above [0190.0.0.23] and [0191.0.0.23] sees for disclosing of [0190.0.0.23] and [0191.0.0.23] these paragraphs [0190.0.0.0] and [0191.0.0.0]

[0191.1.0.23]: see [0191.1.0.0] for disclosing of this paragraph

[0192.0.0.0] was to [0203.0.0.0] above [0192.0.0.23] to [0203.0.0.23] saw for disclosing of [0192.0.0.23] to [0203.0.0.23] these paragraphs

[0204.0.23.23] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding polypeptide or its segmental nucleic acid molecule shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table II B the 5th or 7 row, 275-277 and/or 635 or 636 row of mature form at least, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly Whitfield's ointment quantity increases;

(b) comprise, preferably comprise nucleic acid molecule or its segmental nucleic acid molecule shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row of mature form at least, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by use primer or the nucleic acid molecule of primer to increasing and obtain shown in Table III the 7th row, 275-277 and/or 635 or 636 row from cDNA library or genomic library, and in biological or its part, give each fine chemicals, i.e. the increase of Whitfield's ointment quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like consensus sequence shown in Table IV the 7th row, 275-277 and/or 635 or 636 row and gives each fine chemicals in biological or its part, i.e. the nucleic acid molecule of Whitfield's ointment quantity increase;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, i.e. the nucleic acid molecule that increases of Whitfield's ointment quantity, the structural domain of the polypeptide of described peptide coding shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, nucleic acid molecule shown in 275-277 and/or 635 or 636 row or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 275-277 and/or 635 or 636 row, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from sequence shown in Table I A or IB the 5th or 7 row, 275-277 and/or 635 or 636 row by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention is not made up of the sequence shown in Table I A or IB the 5th or 7 row, 275-277 and/or 635 or 636 row.In another embodiment, nucleic acid molecule of the present invention is identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99% with sequence at least 30% shown in Table I A or IB the 5th or 7 row, 275-277 and/or 635 or 636 row.In another embodiment, the nucleic acid molecule peptide sequence shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row of not encoding.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row at least one or the different polypeptide of a plurality of amino acid, and the protein of shown in Table II A or IIB the 5th or 7 row, 275-277 and/or the 635 or 636 row sequence of therefore not encoding.Therefore, in one embodiment, therefore, in one embodiment, (a) do not form to the coded protein of the nucleotide sequence of (l) by sequence shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row.In another embodiment, protein sequence shown in protein of the present invention and Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row at least 30% identical and with sequence shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row less than 100%, preferably, be more preferably less than 99%, 98%, 97%, 96% or 95% identical less than 99.999%, 99.99% or 99.9%.

Above [0205.0.0.23] and [0206.0.0.23] sees for disclosing of [0205.0.0.23] and [0206.0.0.23] these paragraphs [0205.0.0.0] and [0206.0.0.0]

[0207.0.23.23] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of glutamic acid metabolism, phosphoenolpyruvic acid metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.0] was to [0226.0.0.0] above [0208.0.0.23] to [0226.0.0.23] saw for disclosing of [0208.0.0.23] to [0226.0.0.23] these paragraphs

[0227.0.23.23] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I the 5th or 7 row, 275-277 and/or the 635 or 636 capable sequence or derivatives thereofs of mentioning, can advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes of expressing at least one cinnamate and/or chorismic acid biosynthetic pathway in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with one or more sequences shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.23.23]％

[0229.0.23.23] can relate to the sequence of other gene of cinnamate or chorismic acid biosynthesizing assimilation or catabolic pathway with employed other the favourable nucleotide sequences that the combination of sequence and/or aforementioned biosynthesis gene is expressed shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row of present method for coding.

[0230.0.0.23] sees [0230.0.0.0] for disclosing of this paragraph

[0231.0.23.23] in another advantageous embodiment of the inventive method, the employed biology of present method is to have weakened the salicylic protein of degraded simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.For example it be known to those skilled in the art that, to the inhibition of the salicylic enzyme of degrading or check and to cause that the Whitfield's ointment accumulation increases in the plant.

[0232.0.0.0] was to [0276.0.0.0] above [0232.0.0.23] to [0276.0.0.23] saw for disclosing of [0232.0.0.23] to [0276.0.0.23] these paragraphs

[0277.0.23.23] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.

[0278.0.0.0] was to [0282.0.0.0] above [0278.0.0.23] to [0282.0.0.23] saw for disclosing of [0278.0.0.23] to [0282.0.0.23] these paragraphs

[0283.0.23.23] in addition, can from cell, separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example resist protein shown in Table II the 3rd row, 275-277 and/or 635 or 636 row, the perhaps antibody of polypeptide or its antigen part shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, it can utilize polypeptide of the present invention or its fragment to produce by standard technique.The monoclonal antibody of polypeptide shown in preferably specificity is gone in conjunction with Table II the 5th row, 275-277 and/or 635 or 636 in conjunction with polypeptide shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred specificity.

[0284.0.0.23] sees [0284.0.0.0] for disclosing of this paragraph

[0285.0.23.23] in one embodiment, the present invention relates to have as Table II the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in sequence or by as Table I the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in the coded polypeptide of sequence of nucleic acid molecule or its function homologue.

[0286.0.23.23] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 7th row, consensus sequence shown in 275-277 and/or 635 or 636 row or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 7th row, consensus sequence or by its polypeptide of forming shown in 275-277 and/or 635 or 636 row, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to the polypeptide that comprises more than a consensus sequence (each row) shown in Table IV the 7th row, 275-277 and/or 635 or 636 row.

[0287.0.0.0] was to [0290.0.0.0] above [0287.0.0.23] to [0290.0.0.23] saw for disclosing of [0287.0.0.23] to [0290.0.0.23] these paragraphs

[0291.0.23.23] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from sequence shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, sequence shown in 275-277 and/or 635 or 636 row, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, sequence shown in 275-277 and/or 635 or 636 row.In another embodiment, described polypeptide of the present invention be can't help shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row sequence and is formed.

[0292.0.0.23] sees [0292.0.0.0] for disclosing of this paragraph

[0293.0.23.23] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention has by one or more amino acid and other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row.In another embodiment, polypeptide of the present invention be can't help shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row sequence and is formed.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide is not made up of the coded sequence of nucleic acid molecule shown in Table I A or IB the 5th or 7 row, 275-277 and/or 635 or 636 row.

[0294.0.23.23] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, the polypeptide of activity of proteins shown in 275-277 and/or 635 or 636 row, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, sequence shown in 275-277 and/or 635 or 636 row.

[0295.0.0.0] was to [0297.0.0.0] above [0295.0.0.23] to [0297.0.0.23] saw for disclosing of [0295.0.0.23] to [0297.0.0.23] these paragraphs

[00297.1.0.23]％

[0298.0.23.23] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, 275-277 and/or 635 or 636 the row shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used has and the identical aminoacid sequence of sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row.

[0299.0.23.23] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% with one of nucleotide sequence sequence homology shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises nucleotide sequence or the coded aminoacid sequence of its homologue with nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row.

[0300.0.23.23] therefore, and be described in detail as this paper, and polypeptide of the present invention is because natural variation or mutagenesis can be different with sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row on aminoacid sequence.Therefore, the complete amino acid sequence homology that this polypeptide contains with sequence shown in Table II A or IIB the 5th or 7 row, 275-277 and/or 635 or 636 row is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.23] sees [0301.0.0.0] for disclosing of this paragraph

The biologically-active moiety of [0302.0.23.23] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, the aminoacid sequence shown in 275-277 and/or 635 or 636 row or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.23] sees [0303.0.0.0] for disclosing of this paragraph

[0304.0.23.23] operation nucleic acid molecule of the present invention may cause generation have basically the active of shown in Table II the 3rd row, 275-277 and/or 635 or 636 row polypeptide and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.0] was to [0308.0.0.0] above [0305.0.0.23] to [0308.0.0.23] saw for disclosing of [0305.0.0.23] to [0308.0.0.23] these paragraphs

[0306.1.0.22]％

[0309.0.23.23] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, protein shown in 275-277 and/or 635 or 636 row is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, " other polypeptide " in 275-277 and/or 635 or 636 row is meant the polypeptide of the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, polypeptide shown in 275-277 and/or 635 or 636 row is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, proteinic and shown in 275-277 and/or 635 or 636 row from the protein of identical or different biology.In one embodiment, " other polypeptide " that is not shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row do not given each fine chemicals increase in biology or its part.

[0310.0.0.0] was to [0334.0.0.0] above [0310.0.0.23] to [0334.0.0.23] saw for disclosing of [0310.0.0.23] to [0334.0.0.23] these paragraphs

[0335.0.23.23] confirmed that the dsRNAi method to the expression that reduces nucleotide sequence shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change by the expression that reduces shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row nucleotide sequence and/or its homologue.Double stranded rna molecule at the coded protein expression of the nucleotide sequence that is used for reducing shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.0] was to [0342.0.0.0] above [0336.0.0.23] to [0342.0.0.23] saw for disclosing of [0336.0.0.23] to [0342.0.0.23] these paragraphs

[0343.0.23.23] as describing, in order to cause effective reduction of expression, at dsRNA and shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that sequence or its homologue begin to produce shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row in a kind of organism.

[0344.0.0.0] was to [0361.0.0.0] above [0344.0.0.23] to [0361.0.0.23] saw for disclosing of [0344.0.0.23] to [0361.0.0.23] these paragraphs

[0362.0.23.23] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II the 5th or 7 row, polypeptide shown in 275-277 and/or 635 or 636 row, for example coding has protein such as Table II the 3rd row, the polypeptide of polypeptide active shown in 275-277 and/or 635 or 636 row) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Transgenosis with polypeptide of polypeptide active shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row is meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, the activity that is noted as polypeptide shown in Table II the 3rd row, 275-277 and/or 635 or 636 row (for example having polypeptide of sequence shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row) is enhanced.Example and the inventive method are described in above.

[0363.0.0.23] sees [0363.0.0.0] for disclosing of this paragraph

[0364.0.23.23] when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette--promotor of for example naturally occurring coding gene of polypeptide of the present invention shown in Table II the 3rd row, 275-277 and/or 635 or 636 row and combination of protein coding sequence shown in Table I the 5th row, 275-277 and/or 635 or 636 row accordingly--becomes transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.0] was to [0373.0.0.0] above [0365.0.0.23] to [0373.0.0.23] saw for disclosing of [0365.0.0.23] to [0373.0.0.23] these paragraphs

[0374.0.23.23] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the Whitfield's ointment that produces in the inventive method, particularly each fine chemicals also can be from plant with each fine chemicals of free salicylic acid, particularly free or be bonded to compound or the isolated in form of part (such as but not limited to ester).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

Above [0375.0.0.23] and [0376.0.0.23] sees for disclosing of [0375.0.0.23] and [0376.0.0.23] these paragraphs [0375.0.0.0] and [0376.0.0.0]

[0377.0.23.23] therefore the invention still further relates to the method according to this invention, separated the Whitfield's ointment and/or the salicylate that are produced thus.

[0378.0.23.23] can separate by this way and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than 90% the Whitfield's ointment and/or the salicylate that produce in the method.As required, resultant Whitfield's ointment and/or salicylate can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.23.23] is the mixture of each fine chemicals Whitfield's ointment and/or salicylate by the product that the present invention produced in one embodiment.

The suitable synthetic parent material of Whitfield's ointment that [0380.0.23.23] obtains by carrying out the inventive method as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises Whitfield's ointment that separation produces and salicylate composition or each fine chemicals (if expectation), but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention be the Whitfield's ointment that produces of the inventive method and/or salicylate or genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine purposes or be used to produce the purposes of Whitfield's ointment and/or salicylate, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

Above [0381.0.0.23] and [0382.0.0.23] sees for disclosing of [0381.0.0.23] and [0382.0.0.23] these paragraphs [0381.0.0.0] and [0382.0.0.0]

[0383.0.23.23]％

[0384.0.0.23] sees [0384.0.0.0] for disclosing of this paragraph

The fermented liquid that [0385.0.23.23] obtains with this kind approach particularly contains the fermented liquid with other organic acid, amino acid, polypeptide or polysaccharide blended Whitfield's ointment and/or salicylate, and dry matter content accounts for the 1-70% of weight, preferred 7.5 to 25% usually.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means and to utilize the concentration of sugar during this period of time to remain on or be reduced to 0-10g/l in the fermention medium, preferred to 0-3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0.23.23] therefore can also be further purified the Whitfield's ointment and/or the salicylate that produce according to the present invention.For this reason, the composition that will contain product separates (by for example open column chromatography or HPLC), and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.0] was to [0392.0.0.0] above [0387.0.0.23] to [0392.0.0.23] saw for disclosing of [0387.0.0.23] to [0392.0.0.23] these paragraphs

[0393.0.23.23] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify and the nucleic acid molecule of the present invention nucleic acid molecule of under lax stringent condition, hybridizing of the nucleic acid molecule shown in Table I the 5th or 7 row, 275-277 and/or 635 or 636 row, preferred Table I B the 5th or 7 row, 275-277 and/or 635 or 636 row particularly, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.0] was to [0399.0.0.0] above [0394.0.0.23] to [0399.0.0.23] saw for disclosing of [0394.0.0.23] to [0399.0.0.23] these paragraphs

[00399.1.23.23] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect and whether depends on the active of polypeptide shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row or its homologue or express, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high shown in Table II the 5th or 7 row, 275-277 and/or 635 or 636 row the biology phenotype more much at one of activity of proteins.

[0400.0.0.0] was to [0416.0.0.0] above [0400.0.0.23] to [0416.0.0.23] saw for disclosing of [0400.0.0.23] to [0416.0.0.23] these paragraphs

[0417.0.23.23] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce the biology that the inhibitor of Whitfield's ointment biosynthetic pathway is had resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block Whitfield's ointment synthetic inhibitor.

In addition, express as the crossing of Table I the 5th and 7 row, 275-277 and/or 635 or 636 nucleotide sequences that characterized of row and increased the salicylic acid content in the vegetable cell and also can be used for the tolerance of plant nickel (Ni)/zinc (Zn).

[0418.0.0.0] was to [0423.0.0.0] above [0418.0.0.23] to [0423.0.0.23] saw for disclosing of [0418.0.0.23] to [0423.0.0.23] these paragraphs

[0424.0.23.23] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other organic acids.Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.0] was to [0435.0.0.0] above [0425.0.0.23] to [0435.0.0.23] saw for disclosing of [0425.0.0.23] to [0435.0.0.23] these paragraphs

[0436.0.23.23] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement the mutagenesis in vivo of yeast belong, genus mortierella, Escherichia and above-mentioned other genus, be beneficial to produce Whitfield's ointment.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.23.23] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.23.23] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.23.23] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.23.23] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad 50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.0] was to [0443.0.0.0] above [0441.0.0.23] to [0443.0.0.23] saw for disclosing of [0441.0.0.23] to [0443.0.0.23] these paragraphs

[0444.0.23.23] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.23.23] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.0] was to [0453.0.0.0] above [0446.0.0.23] to [0453.0.0.23] saw for disclosing of [0446.0.0.23] to [0453.0.0.23] these paragraphs

[0454.0.23.23] embodiment 8: the influence that analyzing nucleic acid molecules produces Whitfield's ointment

[0455.0.23.23] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example Whitfield's ointment and/or salicylate) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being Whitfield's ointment and/or salicylate) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia ofIndustrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry andMolecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recoveryand purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", JohnWiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recoveryprocesses for biological Materials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques inbiotechnology ", Noyes Publications).

[0456.0.0.23]: see [0456.0.0.0] for disclosing of this paragraph

[0457.0.23.23] embodiment 9: salicylic purifying

[0458.0.23.23] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS, GC-MS or TLC can clearly detect salicylic existence to the biology analysis of recombinating.

It is biological to use the following step to analyze, as the total amount that produces in the inventive method used yeast: by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 402 2S1V diluters with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.20ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, used Hewlett Packard 1100HPLC, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Technologies, can be from Ultra Scientific Inc., 250Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6x 250mm[Waters, 34Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

[0459.0.23.23] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RTHPLC to be further purified Whitfield's ointment.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.0.23] sees [0460.0.0.0] for disclosing of this paragraph

[0461.0.23.23] embodiment 10: clone SEQ ID NO:33502,33810,33899,98917 or 99068 is used for expressing plant

[0462.0.0.23] sees [0462.0.0.0] for disclosing of this paragraph

[0463.0.23.23] passes through pcr amplification SEQ ID NO:33502,33810,33899,98917 or 99068 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.0] was to [0466.0.0.0] above [0464.0.0.23] to [0466.0.0.23] saw for disclosing of [0464.0.0.23] to [0466.0.0.23] these paragraphs

[0466.1.0.23] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.23.23] selects following primer sequence for genes of SEQ ID NO:33502:

I) forward primer (SEQ ID NO:33802)

atgaaaaaaa?ccacattagc?actg

Ii) reverse primer (SEQ ID NO:33803)

ttactgcatt?aacaggtaga?tggtg

Select following primer sequence for genes of SEQ ID NO:33810:

I) forward primer (SEQ ID NO:33894)

atgatcagga?gtcacaccat?ga

Ii) reverse primer (SEQ ID NO:33895)

ttaattgcca?gccatcgcct?g

Select following primer sequence for genes of SEQ ID NO:33899:

I) forward primer (SEQ ID NO:34217)

atgttatcac?tgtctgccaa?aaatc

Ii) reverse primer (SEQ ID NO:34218)

tcagtcacgg?tattggtcaa?aaaat

Select following primer sequence for genes of SEQ ID NO:98917:

I) forward primer (SEQ ID NO:99063)

atggaaacga?ctcaaaccag?cac

Ii) reverse primer (SEQ ID NO:99064)

ttagctgaac?agagagtaga?agatt

Select following primer sequence for genes of SEQ ID NO:99068:

I) forward primer (SEQ ID NO:99168)

atgagtactt?cagatagcat?tgtatc

Ii) reverse primer (SEQ ID NO:99169)

ttaaaacagt?ttgtatacga?tgttcag

[0468.0.0.0] was to [0470.0.0.0] above [0468.0.0.23] to [0470.0.0.23] saw for disclosing of [0468.0.0.23] to [0470.0.0.23] these paragraphs

The PCR product that [0470.1.23.23] utilizes the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBIFermentas) phosphorylation to produce by Pfu Turbo archaeal dna polymerase, and the clone enters in the binary vector of treated mistake.

[0471.0.23.23] sees [0471.0.0.0] for disclosing of this paragraph

[0471.1.23.23] uses Pfu Turbo archaeal dna polymerase benefit flat in the second step building-up reactions by the DNA end of the PCR product that the Herculase archaeal dna polymerase produces.The composition of schedule of operation of mending flat DNA end is as follows: 0.2mM mends flat dTTP and 1.25u Pfu Turbo archaeal dna polymerase.Being reflected at 72 ℃ hatched 30 minutes.Utilize the T4DNA polysaccharase and use Standard operation procedure SOP (for example MBI Fermentas) phosphorylation PCR product, and be cloned in the carrier of handling.

[0472.0.0.0] was to [0479.0.0.0] above [0472.0.0.23] to [0479.0.0.23] saw for disclosing of [0472.0.0.23] to [0479.0.0.23] these paragraphs

[0480.0.23.23] embodiment 11: express the generation of SEQ ID NO:33502,33810,33899,98917 or 99068 transgenic plant

[0482.0.0.0] was to [0513.0.0.0] above [0481.0.0.23] to [0513.0.0.23] saw for disclosing of [0481.0.0.23] to [0513.0.0.23] these paragraphs

[0514.0.23.23] alternatively, as Kmetec, V., J.Pharm.Biomed.Anal.1992Oct-Dec; 10 (10-12): detect Whitfield's ointment described in the 1073-6.

The different plants of being analyzed the results are shown in following table 1:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??b0161	Whitfield's ointment	??LC	??1.67	??5.75
??b2664	Whitfield's ointment	??LC	??1.56	??5.25
					??YLR089C	Whitfield's ointment	??LC	??1.45	??2.53
??b2796	Whitfield's ointment	??LC	??1.42	??3.48
					??b3116	Whitfield's ointment	??LC	??1.41	??2.30

[0515.0.23.23] the 2nd row have shown the metabolite Whitfield's ointment of being analyzed.The 4th row and the 5th row have shown the ratio of metabolite between transgenic plant and wild-type of being analyzed.Metabolite increases: maximum value: maximum x-is (to the wild-type stdn)-minimum value doubly: minimum x-is (to the wild-type stdn) doubly.Metabolite reduces: maximum value: doubly (to the wild-type stdn) (bottom line minimizing) of maximum x-, minimum value: minimum x-is (to the wild-type stdn) (reducing) to greatest extent doubly.The 3rd row have been pointed out analytical procedure.

[0516.0.0.0] was to [0530.0.0.0] above [0516.0.0.23] to [0530.0.0.23] saw for disclosing of [0516.0.0.23] to [0530.0.0.23] these paragraphs

[0530.1.0.0] was to [0530.6.0.0] above [0530.1.0.23] to [0530.6.0.23] saw for disclosing of [0530.1.0.23] to [0530.6.0.23] these paragraphs

[0531.0.0.0] was to [0552.0.0.0] above [0531.0.0.23] to [0552.0.0.23] saw for disclosing of [0531.0.0.23] to [0552.0.0.23] these paragraphs

[0552.1.0.23]：％

[0552.2.0.23] sees [0552.2.0.0] for disclosing of this paragraph

[0553.0.23.23]

1. produce salicylic method, it comprises:

(a) in the activity that improves or produce shown in Table II the 5th or 7 row, 275-277 or 635 and 636 row protein or its function equivalent in non-human being or its one or more parts; With

(b) in allowing described biology, produce this biology of cultivation under the salicylic condition.

2. produce salicylic method, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, 275-277 or 635 and 636 row, and described nucleic acid molecule is given Whitfield's ointment quantity increase in biology or its part;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row, 275-277 or 635 and 636 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of Whitfield's ointment quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of Whitfield's ointment quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that Whitfield's ointment quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 275-277 or 635 and 636 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives that Whitfield's ointment quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that Whitfield's ointment quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 275-277 or 635 and 636 row and give biology or its part in Whitfield's ointment quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give Whitfield's ointment quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded Whitfield's ointment.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by the free or the bonded Whitfield's ointment of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, 275-277 or 635 and 636 row, and described nucleic acid molecule is given Whitfield's ointment quantity increase in biology or its part;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row, 275-277 or 635 and 636 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of Whitfield's ointment quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of Whitfield's ointment quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that Whitfield's ointment quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row, 275-277 or 635 and 636 row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives that Whitfield's ointment quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that Whitfield's ointment quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row, 275-277 or 635 and 636 row and give biology or its part in Whitfield's ointment quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give Whitfield's ointment quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from sequence shown in Table I A the 5th or 7 row, 275-277 or 635 and 636 row by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, it produces by the method described in claim 13 or by the nucleic acid molecule encoding described in claim 6, this polypeptide is different from sequence shown in Table II A the 5th or 7 row, 275-277 or 635 and 636 row by one or more amino acid thus.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in Whitfield's ointment quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part Whitfield's ointment quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps Whitfield's ointment level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the Whitfield's ointment level that will measure or expression of polypeptides level and standard Whitfield's ointment or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that the Whitfield's ointment generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of Whitfield's ointment quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of Whitfield's ointment quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that the Whitfield's ointment generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that Whitfield's ointment increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the suitable salicylic host cell that produces;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) the Whitfield's ointment level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give Whitfield's ointment level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that the Whitfield's ointment generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that Whitfield's ointment quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the suitable salicylic host cell that produces;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) the Whitfield's ointment level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give Whitfield's ointment level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that Whitfield's ointment increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control Whitfield's ointment level at biology.

25. agrochemicals, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of any one described method of claim 1-5, claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein fine chemicals is a Whitfield's ointment.

27. according to claim 10-12A any one described host cell or plant, its anti-biosynthetic weedicide of Whitfield's ointment that suppresses.

28. according to claim 10-12A any one described host cell or plant, it demonstrates the antiviral resistance of comparing raising with control plant.

29. according to claim 10-12A any one described host cell or plant, it demonstrates the resistance of the antibiont or the abiotic stress of raising.

30. according to claim 10-12A any one described host cell or plant, it demonstrates old and feeble the reduction.

31. the host cell of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it makes the anti-Whitfield's ointment synthetic weedicide that suppresses of plant.

[0554.0.0.23] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.24] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.0] seen in disclosing of [0001.0.0.24] these paragraphs

Disclosing of [0002.0.24.24] to [0009.0.24.24] these paragraphs sees that [0002.0.24.24] is to [0009.0.10.10]

The quality that [0010.0.24.24] therefore improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the β-Hu Luobusu that for example is present in as mentioned above in plant and some microorganisms is limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is carotenoid spectrum in the balanced diet as far as possible, because excessive carotenoid has negative effect on the specific concentrations.Further improving the quality only may be by adding other carotenoid, and these compositions all are limited.

[0011.0.10.10] seen in disclosing of [0011.0.24.24] these paragraphs

[0012.0.24.24] therefore, still be starved of one or more codings and participate in carotenoid (for example β-Hu Luobusu or its precursor such as isopentenylpyrophosphate (IPP)) biosynthetic enzyme or other proteinic suitable gene, and make and not form unwanted byproduct with specific these products of generation of technical scale.

Be used for the screening of biosynthetic gene, above two specific all be particularly important.On the one hand, but forever need to improve the method that obtains the highest intrinsic energy carotenoid such as β-Hu Luobusu; On the other hand, reduce the byproduct that produces in the production process as far as possible.

[0013.0.0.0] seen in disclosing of [0013.0.0.24] these paragraphs

[0014.0.24.24] therefore in the first embodiment, the present invention relates to produce the method for fine chemicals, and fine chemicals is carotene or its precursor thus.In preferred embodiments, fine chemicals is β-Hu Luobusu or IPP.Therefore, in the present invention, as used herein term " fine chemicals " refers to " carotene ", particularly β-Hu Luobusu or its precursor, particularly IPP.In addition, as used herein term " fine chemicals " refers to comprise the fine chemicals of carotene, particularly β-Hu Luobusu or its precursor, particularly IPP.

[0015.0.24.24] in one embodiment, term " carotene, particularly β-Hu Luobusu " or " fine chemicals " or " each fine chemicals " meaning is meant at least a have carotene, the particularly active chemical compound of β-Hu Luobusu sample.

In preferred embodiments, term " carotene precursor; the IPP particularly " meaning is meant chemical compound, preferably, its expression makes carotene in the biology, preferably beta-carotene activity or quantity (separately or with other chemical compound) increase, for example other representation aids is as giving the gene of IPP conversion reaction or generation IPP, for example participate in mevalonic acid (the special precursor of first of terpene) from acetyl-CoA via the HMG-CoA (gene that 3-hydroxy-3-methyl glutaryl-CoA) produces or transforms, mevalonic acid self is converted to isopentenylpyrophosphate (IPP) or geranylpyrophosphate (GGPP) molecule, it produces colourless phytoene, and phytoene is initial carotenoid; The already present mevalonic acid that optionally forms for IPP does not rely on approach, and (it is the plastid sign of green alga and higher plant at first several eubacteriums; The transketolase type condensation reaction that comprises pyruvic acid and D-glyceraldehyde-3-phosphate is to obtain 1-deoxy-D-xylulose-5-phosphoric acid (DXP)); Or one of desaturase, its carry out a series of desaturation reaction (with the IPP phytoene be transformed into phytofluene ,-carotene, neurosporene and final Lyeopene) or β-Hu Luobusu (comprising two β-ionene ring) carried out cyclization.

In one embodiment, term " fine chemicals " meaning is meant " carotene, particularly β-Hu Luobusu and precursor thereof, particularly IPP ".In one embodiment, according to the context that uses it, term " fine chemicals " meaning is meant β-Hu Luobusu or IPP.In whole specification sheets, term " fine chemicals " dissociate fine chemicals, its salt, ester, thioesters or be bonded to other compound such as the form of sugar or glycopolymers (as glucoside, for example diglucoside).In one embodiment, term " fine chemicals " meaning is meant free form or its salt form or its ester-formin or is bonded to the β-Hu Luobusu or the IPP of glucoside (for example diglucoside).

[0016.0.24.24] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b1829, b2699, YBR089C-A, YDR316W, YDR513W, and/or YLL013C, and/or b0730, b1926, b2211, b3172, b4129, and/or YDR407C and/or b0481, b0970, b1736, b1738 and/or b3160 protein; With

(b) in allowing described biology, produce under the condition that fine chemicals is carotene, particularly β-Hu Luobusu or its precursor, particularly IPP and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein have Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in activity of proteins, perhaps have by Table I A or IB the 5th or 7 row, 278-289 is capable and/or 637-641 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce fine chemicals be carotene, particularly β-Hu Luobusu or its precursor, particularly IPP or comprise carotene, particularly β-Hu Luobusu or the condition of the fine chemicals of its precursor, particularly IPP under make biological growth.

[0016.1.24.24] therefore, term " fine chemicals " meaning is meant and Table I-IV the 279th, 282,285,286,288 and/or 289 row and/or relevant " β-Hu Luobusu " or its homologues of the capable listed full sequence of 637-641 in one embodiment, and looks like and be meant and relevant " IPP " or its homologue of full sequence shown in Table I-IV the 278th, 280,281,283,284 and/or 287 capable and/or 637-641 are capable.Therefore, term " fine chemicals " can refer to " β-Hu Luobusu " or " IPP " according to environment and context.For being meant, the meaning of illustrating term " each fine chemicals " " β-Hu Luobusu " and/or " IPP " from the listed sequence of context can also use term " each fine chemicals ".

[0017.0.0.24] to [0018.0.0.24]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.24.24] produces each fine chemicals advantageously causes for example generation enhancing of IPP of carotene, particularly β-Hu Luobusu or its precursor.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table I A or IB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, protein active shown in 284 and/or 287 row and/or 637-641 are capable or by as Table I A or IB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in 284 and/or 287 row and/or 637-641 were capable.

[0020.0.24.24] is surprisingly found out that, β-Hu Luobusu (with regard to described protein and homologue and coding nucleic acid molecule (particularly shown in Table I-IV the 3rd row, the 279th, 282,285,286,288 and/or 289 row), being " fine chemicals " or " each fine chemicals ") content that e. coli k12 or yeast saccharomyces cerevisiae protein b1829, b2699, YBR089C-A, YDR316W, YDR513W and YLL013C give the plant that transforms at transgene expression in the Arabidopis thaliana increases.

Be surprisingly found out that, e. coli k12 or yeast saccharomyces cerevisiae protein b0730, b1926, b2211, b3172, b4129, or IPP (with regard to described protein and homologue and coding nucleic acid molecule (particularly as shown in table 1-IV the 3rd row, the 278th, 280,281,283,284 or 287 row or 637-641 are capable), being " fine chemicals " or " each fine chemicals ") content that YDR407C and/or b0481, b0970, b1736, b1738 and/or b3160 give the plant that transforms at transgene expression in the Arabidopis thaliana increases.

[0021.0.0.0] seen in disclosing of [0021.0.0.24] these paragraphs

The sequence of [0022.0.24.24] e. coli k12 b0730 has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be that succinic thiokinase operon transcriptional and acyl are replied regulatory gene.Therefore, in one embodiment, the inventive method comprises transcriptional GntR superfamily protein as shown here, particularly participate in C-compound and carbohydrate utilization, transcribe control, protokaryon Nucleotide, transcription repressor, DNA is in conjunction with the protein of regulating, preferred succinic thiokinase operon transcriptional and acyl are replied the purposes of regulatory gene or its homologue, it is used for producing each fine chemicals in biological or its part as described, be IPP or carotene, the IPP or the carotene of preferably free or combining form.In one embodiment, in the methods of the invention, succinic thiokinase operon transcriptional and acyl are replied regulatory gene, particularly the activity of the lipoprotein of transcriptional GntR superfamily is enhanced or produces, for example reply regulatory gene from colibacillary succinic thiokinase operon transcriptional and acyl, particularly the activity of the lipoprotein of transcriptional GntR superfamily or its homologue is enhanced or produces.

The sequence of e. coli k12 b1829 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the heat shock protein with protease activity.Therefore, in one embodiment, the inventive method comprises heat shock protein htpX superfamily protein as shown here, particularly participate in coercing the protein of reaction, unclassified protein, pheromone reaction, mating type decision, sex-specific protein, protein modification, proteolytic degradation, the purposes that preferably has heat shock protein or its homologue of protease activity, it is used for producing each fine chemicals in biological or its part as described, be carotene, particularly β-Hu Luobusu preferably dissociates or combining form.In one embodiment, in the methods of the invention, the activity that described activity for example has the heat shock protein of protease activity is enhanced or produces, and for example has the heat shock protein of protease activity or the activity of its homologue is enhanced or produces from colibacillary.

The sequence of e. coli k12 b1926 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be flagellin matter fliT.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary flagellin matter fliT or its homologue as shown here, and it is used for producing each fine chemicals in biological or its part as described, be IPP or carotene, preferably free or combining form.In one embodiment, in the methods of the invention, the described activity for example activity of flagellin matter fliT is enhanced or produces, and for example the activity from colibacillary flagellin matter fliT or its homologue is enhanced or produces.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be DNA chain exchange and the recombinant protein with recombinant protein recA superfamily proteolytic enzyme and nuclease.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary recombinant protein recA superfamily protein, preferably have DNA reorganization and DNA repairing activity, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, particularly have the DNA chain exchange of proteolytic enzyme and nuclease and the purposes of recombinant protein or its homologue, it is used for producing each fine chemicals in biological or its part as described, be carotene, particularly β-Hu Luobusu preferably dissociates or combining form.In one embodiment, in the methods of the invention, described activity is proteolytic enzyme and nuclease for example, particularly have the DNA chain exchange of proteolytic enzyme and nuclease and the activity of recombinant protein, particularly the activity of recombinant protein recA superfamily is enhanced or produces, and for example the activity from colibacillary these protein or its homologue is enhanced or produces.

The sequence of e. coli k12 b2211 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be ATP-binding cassette superfamily ATP-binding transport albumen.Therefore, in one embodiment, the inventive method comprises as shown here from the colibacillary ATP-binding cassette superfamily protein of not specifying, preferably has Nucleotide combination, abc transport body, cell traffic and the active protein of transporting mechanism, the purposes of ATP-binding cassette superfamily ATP-binding transport albumen or its homologue particularly, it is used for producing each fine chemicals in biological or its part as described, and promptly IPP or carotene preferably dissociate or combining form.In one embodiment, in the methods of the invention, the described activity for example proteic activity of ATP-binding cassette superfamily ATP-binding transport is enhanced or produces, and for example the activity from colibacillary ATP-binding cassette superfamily ATP-binding transport albumen or its homologue is enhanced or produces.

The sequence of e. coli k12 b3172 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be arginyl succsinic acid synthetic enzyme.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary arginyl succsinic acid synthase superfamily protein, the preferred amino acid bio that participates in synthesizes, nitrogen and sulfo-are thanked, L-glutamic acid family (proline(Pro), oxyproline, arginine, glutamine, L-glutamic acid) biosynthesizing, L-glutamic acid family amino acid degradation, nitrogen and sulphur utilization, ornithine cycle, polyamines and sarkosine biosynthesizing, the aspartate family biosynthesizing, the ammonia assimilation effect, the biosynthetic protein of L-glutamic acid family, preferably have the protein of arginyl succsinic acid synthase activity or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be IPP or carotene, preferably free or combining form.In one embodiment, in the methods of the invention, the described activity for example activity of arginyl succsinic acid synthetic enzyme is enhanced or produces, and for example the activity from colibacillary arginyl succsinic acid synthetic enzyme or its homologue is enhanced or produces.

The sequence of e. coli k12 b4129 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be derivable Methionin tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises as shown here from colibacillary Lysine-tRNA ligase superfamily protein, the preferred protein that participates in aminoacyl-tRNA-synthetic enzyme translation, preferably have the protein of derivable Methionin tRNA synthase activity or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, and promptly IPP or carotene preferably dissociate or combining form.In one embodiment, in the methods of the invention, the activity of for example derivable Methionin tRNA of described activity synthetic enzyme is enhanced or produces, and for example the activity from colibacillary derivable Methionin tRNA synthetic enzyme or its homologue is enhanced or produces.

The sequence of yeast saccharomyces cerevisiae YBR089C-A has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be nonhistones chromosomal protein.Therefore, in one embodiment, the inventive method comprises NHC protein HMG-2 as shown here, HMG box homology, does not specify HMG box protein matter superfamily protein, excellent participation is transcribed control, nucleic acid in conjunction with, the protein of transcribing, the purposes of preferred NHC protein or its homologue, it is used for producing each fine chemicals in biological or its part as described, be carotene, β-Hu Luobusu particularly particularly increases the quantity of free or combining form.In one embodiment, in the methods of the invention, the described activity for example activity of NHC protein is enhanced or produces, and for example is enhanced or produces from the NHC protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YDR316W has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity be defined as be the supposition seven β-thigh family S-adenosylmethionine dependency methyltransgerase.Therefore, in one embodiment, the inventive method comprises bioC homology superfamily protein as shown here, seven β-thigh family S-adenosylmethionine dependency methyltransgerase of preferred supposition or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be carotene, β-Hu Luobusu particularly particularly increases the quantity of free or combining form.In one embodiment, in the methods of the invention, the activity of seven β that described activity is for example supposed-thigh family S-adenosylmethionine dependency methyltransgerase is enhanced or produces, and for example is enhanced or produces from seven β-thigh family S-adenosylmethionine dependency methyltransgerase of the supposition of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YDR407C has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be target complex body (TRAPP) component that participates in the transhipment of ER-Golgi membrane.Therefore, in one embodiment, the inventive method comprises the protein of participation the vesica transhipment (Golgi network etc.) as shown here, the preferred purposes that participates in target complex body (TRAPP) component of ER-Golgi membrane transhipment, it is used for producing each fine chemicals in biological or its part as described, be IPP or carotene, particularly increase the quantity of free or combining form.In one embodiment, in the methods of the invention, described activity for example vesica transhipment (Golgi network etc.) activity of proteins is enhanced or produces, and for example is enhanced or produces from vesica transhipment (Golgi network etc.) protein of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of yeast saccharomyces cerevisiae YDR513W has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be glutathione reductase.Therefore, in one embodiment, the inventive method comprises glutaredoxin superfamily protein as shown here, particularly participate in deoxyribonucleotide metabolism, cytoplasm, coerce the protein that the relevant energy of reaction, detoxifcation, electron transport and film is preserved, the purposes of preferred glutathione reductase or its homologue, it is used for producing each fine chemicals in biological or its part as described, be carotene, β-Hu Luobusu particularly particularly increases the quantity of free or combining form.In one embodiment, in the methods of the invention, the described activity for example activity of glutathione reductase is enhanced or produces, and for example is enhanced or produces from the glutathione reductase of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of YLL013C yeast saccharomyces cerevisiae has been published in Jacq etc., Nature 387 (6632 supplementary issue), 75-78,1997, and Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be PUF protein families (it is from the member pumilio that found and Fbf name) member.Therefore, in one embodiment, the inventive method comprises participation cytodifferentiation as shown here, growth (phylogeny), nucleic acid combination, transcribes the protein of control, other transcriptional activity, preferred PUF protein families (its member pumilio and Fbf name) member from being found, or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be carotene, β-Hu Luobusu particularly particularly increases the quantity of free or combining form.In one embodiment, in the methods of the invention, described activity for example PUF protein families (its member pumilio and Fbf name from being found) member's activity is enhanced or produces, and for example is enhanced or produces from PUF protein families (its member pumilio and Fbf name from the being found) member of yeast saccharomyces cerevisiae or the activity of its homologue.

The sequence of e. coli k12 b0481 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its protein to be defined as be the protein with YbaK-spline structure territory.Therefore, in one embodiment, the inventive method comprises as shown here from the protein in the colibacillary YbaK-of having spline structure territory or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be IPP, the IPP of preferably free or combining form.In one embodiment, in the methods of the invention, the activity of proteins in the described YbaK-of having spline structure territory is enhanced or produces, and for example is enhanced or produces from the protein in the colibacillary YbaK-of having spline structure territory or the activity of its homologue.

The sequence of e. coli k12 b0970 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be glutamate receptor.Therefore, in one embodiment, the inventive method comprises from colibacillary having an active gene product of intestinal bacteria ybhL protein superfamily, the purposes that preferably has the active protein of glutamate receptor protein matter or its homologue, it is used for producing fine chemicals in biological or its part as described, be glutamine and/or isopentenylpyrophosphate, particularly increase glutamine and/or isopentenylpyrophosphate, preferably free or the glutamine of combining form and/or the quantity of isopentenylpyrophosphate.

The sequence of e. coli k12 b1736 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its protein is defined as having the protein of PEP dependency phosphate transferase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein of the colibacillary PEP of having dependency phosphate transferase activity or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be IPP, the IPP of preferably free or combining form.In one embodiment, in the methods of the invention, the activity of PEP dependency phosphotransferase is enhanced or produces, and for example the activity from colibacillary PEP dependency phosphotransferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b1738 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its protein is defined as having the protein of PEP dependency phosphate transferase activity.Therefore, in one embodiment, the inventive method comprises as shown here from the protein of the colibacillary PEP of having dependency phosphate transferase activity or the purposes of its homologue, it is used for producing each fine chemicals in biological or its part as described, be IPP, the IPP of preferably free or combining form.In one embodiment, the activity of PEP dependency phosphotransferase is enhanced or produces in the methods of the invention, and for example the activity from colibacillary PEP dependency phosphotransferase or its homologue is enhanced or produces.

The sequence of e. coli k12 b3160 has been published in Blattner etc., Science277 (5331), and 1453-1474,1997, and its protein is defined as having luciferase-the monooxygenase protein of sample atpase activity.Therefore, in one embodiment, the inventive method comprises as shown here having the monooxygenase protein of luciferase-sample atpase activity or a purposes of its homologue from colibacillary, it is used for producing each fine chemicals in biological or its part as described, be IPP, the IPP of preferably free or combining form.In one embodiment, in the methods of the invention, monooxygenase activity of proteins with luciferase-sample atpase activity is enhanced or produces, and for example has the monooxygenase protein of luciferase-sample atpase activity or the activity of its homologue is enhanced or produces from colibacillary.

The homologous compound (=homologue) of [0023.0.24.24] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, any one homologue of polypeptide shown in Table II A or IIB the 5th or 7 row, the 287th row be have same or similar active shown in Table II A or IIB the 3rd row, the 287th row the homologue of polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferred IPP content increases, and also gives carotene, particularly content beta-carotene increase in other embodiment preferred.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 287th row.In one embodiment, the homologue of one of polypeptide shown in Table II A or IIB the 3rd row, the 287th row is from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 287th row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 287th row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 287th row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 287th row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 287th row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 287th row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II A or IIB the 5th or 7 row, the 278th, 280,281,283,284 row or 637-641 capable shown in the homologue of one of polypeptide be to have same or similar active homologue as polypeptide shown in Table II A or IIB the 3rd row, the 278th, 280,281,283,284 row or 637-641 are capable.Particularly, each fine chemicals in the biology is given in active raising, preferred IPP content increases, and more preferably also giving carotene, particularly content beta-carotene increases.In one embodiment, homologue be have Table I or II the 7th row, the 278th, 280,281,283,284 row or 637-641 capable shown in the homologue of sequence.In one embodiment, Table II A or IIB the 3rd row, the 278th, 280,281,283,284 row or 637-641 capable shown in the homologue of one of polypeptide from bacterium.In one embodiment, Table II A or IIB the 3rd row, the 278th, 280,281,283,284 row or 637-641 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II A or IIB the 3rd row, the 278th, 280,281,283,284 row or 637-641 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II A or IIB the 3rd row, the 278th, 280,281,283,284 row or 637-641 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II A or IIB the 3rd row, the 278th, 280,281,283,284 row or 637-641 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 278th, 280,281,283,284 row or 637-641 are capable is to have same or similar activity (particularly, active raising give IPP content increases in biology or its part) and from the homologue of Escherichia.

In one embodiment, the homologue of one of polypeptide shown in Table II A or IIB the 5th or 7 row, the 285th, 286,288 or 289 row be have same or similar active shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row the homologue of polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferably beta-carotene content increases.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 285th, 286,288 or 289 row.In one embodiment, the homologue of one of polypeptide shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row is from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row is from yeast.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row is from the yeast guiding principle.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row is the homologue from Saccharomycetes.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row is to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 285th, 286,288 or 289 row is to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, the homologue of one of polypeptide shown in Table II A or IIB the 5th or 7 row, the 279th or 282 row be have same or similar active shown in Table II A or IIB the 3rd row, the 279th or 282 row the homologue of polypeptide.Particularly, each fine chemicals in the biology is given in active raising, preferably beta-carotene content increases.In one embodiment, homologue is the homologue with sequence shown in Table I or II the 7th row, the 279th or 282 row.In one embodiment, the homologue of one of polypeptide shown in Table II A or IIB the 3rd row, the 279th or 282 row is from bacterium.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 279th or 282 row is from Proteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 279th or 282 row is to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 279th or 282 row is from the enterobacteria order.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 279th or 282 row is the homologue from enterobacteriaceae.In one embodiment, the homologue of polypeptide shown in Table II A or IIB the 3rd row, the 279th or 282 row or 637-641 are capable is to have same or similar activity (particularly, active raising give content beta-carotene increases in biology or its part) and from the homologue of Escherichia.

[0023.1.24.24] Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the homologue of polypeptide shown in 284 and/or 287 row and/or 637-641 are capable can be by Table I A or IB the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide of nucleic acid molecule encoding or Table II A shown in 284 and/or 287 row and/or 637-641 are capable or IIB the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide shown in 284 and/or 287 row and/or 637-641 are capable.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row can be have increase content beta-carotene and/or quantity is active, by polypeptide shown in Table I A or IB the 7th row, the polypeptide that is respectively nucleic acid molecule encoding shown in the 279th, 282,285,286,288 and/or 289 row or Table II A or IIB the 7th row, the 279th, 282,285,286,288 and/or 289 row.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row can be have increase content beta-carotene and/or quantity is active, by polypeptide shown in the polypeptide of nucleic acid molecule encoding shown in Table I A or IB the 7th row, the 279th, 282,285,286,288 and/or 289 row or Table II A or IIB the 7th row, the 279th, 282,285,286,288 and/or 289 row.

Table II A or IIB the 3rd row, the 278th, 280,281,283, the homologue of polypeptide shown in 284 and/or 287 row and/or 637-641 are capable can be have the IPP content of increasing and/or quantity active, by Table I A or IB the 7th row, be respectively the 278th, 280,281,283, the polypeptide of nucleic acid molecule encoding or Table II A shown in 284 and/or 287 row and/or 637-641 are capable or IIB the 7th row, the 278th, 280,281,283, polypeptide shown in 284 and/or 287 row and/or 637-641 are capable.

The homologue of polypeptide shown in Table II A or IIB the 3rd row, the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable can be have increase IPP content and/or quantity is active, by Table I A or IB the 7th row, the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the polypeptide of nucleic acid molecule encoding or Table II A or IIB the 7th row, the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in polypeptide.

[0024.0.0.0] seen in disclosing of [0024.0.0.24] these paragraphs

[0025.0.24.24] is according to the present invention, the β-Hu Luobusu level increases in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have activity of proteins shown in Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row.In preferred embodiments, protein or polypeptide have proteinic above-mentioned extra activity shown in Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row.In this manual, if this kind protein or polypeptide still have Table II A or IIB the 3rd row, the 279th, 282,285,286, any one proteinic biology or the enzyme activity shown in 288 and/or 289 row, if promptly with Table II A or IIB the 3rd row, the 285th, 286, yeast saccharomyces cerevisiae protein or Table II A shown in 288 or 289 row or IIB the 3rd row, shown in the 279th or 282 row in the e. coli k12 protein any one compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

According to the present invention, the IPP level increases in biology or its part, the preferred described biomass cells if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause, then described protein or polypeptide have " activity of proteins of the present invention ", for example promptly have Table II A or IIB the 3rd row, the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II A or IIB the 3rd row, the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II A or IIB the 3rd row, the 278th, 280,281,283, any one proteinic biology or the enzyme activity shown in 284 and/or 287 row and/or 637-641 are capable, if promptly with Table II A or IIB the 3rd row, yeast saccharomyces cerevisiae protein or Table II A shown in the 287th row or IIB the 3rd row, the 278th, 280,281, shown in 283 and/or 284 row and/or 637-641 are capable in the e. coli k12 protein any one compared, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

In one embodiment, if its next leisure is evolved upward close with biology shown in Table I the 4th row and expressed in evolution with in the eozoan biology far away, polypeptide of the present invention is still given described activity, as each fine chemicals of increase in biological or its part.It is biological from different sections, order, guiding principle or door for example to play eozoan and expression, and plays biology shown in eozoan and Table I the 4th row from identical section, order, guiding principle or door.

[0025.1.0.24]see[0025.1.0.0]

[0026.0.0.24] to [0033.0.0.24]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.24.24] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, it has Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, protein shown in 284 and/or 287 row and/or 637-641 are capable, or by Table I A or IB the 5th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the protein of nucleic acid molecule encoding shown in 284 and/or 287 row and/or 637-641 are capable, or its homologue is (as Table I A or IB the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, homologue shown in 284 and/or 287 row and/or 637-641 are capable) active protein expression level or active aspect difference, and its difference aspect biological chemistry or genetics reason.Therefore it shows each fine chemicals quantity that increases.

[0035.0.0.24] to [0038.0.0.24]: see that [0035.0.0.0] is to [0038.0.0.0]

[0039.0.0.0] seen in disclosing of [0039.0.0.24] these paragraphs

[0040.0.0.24] to [0044.0.0.24]: see that [0040.0.0.0] is to [0044.0.0.0]

[0045.0.24.24] is at e. coli k12 protein b0730 or its homologue, for example succinic thiokinase operon transcriptional and acyl are replied under the situation that the activity of regulatory gene (for example shown in Table II A or IIB the 5th or 7 row, the 278th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 41% and 115% or more between.

In one embodiment, at e. coli k12 protein b1829 or its homologue, for example have under the situation that the activity of the heat shock protein (for example shown in Table II A or IIB the 5th or 7 row, the 279th row) of protease activity is enhanced, preferably, give in one embodiment each fine chemicals, preferably beta-carotene be increased in 33% and 74% or more between.

In one embodiment, at e. coli k12 protein b1926 or its homologue, for example under the situation that the activity of flagellin matter fliT (for example shown in Table II A or IIB the 5th or 7 row, the 280th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 37% and 55% or more between.

In one embodiment, at e. coli k12 protein b2211 or its homologue, for example under the situation that the activity of ATP-binding cassette superfamily ATP-binding transport albumen (for example shown in Table II A or IIB the 5th or 7 row, the 281st row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 46% and 48% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example DNA recombinates and the DNA repairing activity, the pheromone reactive behavior, the mating type decision is active, the sex-specific protein active, Nucleotide is in conjunction with the protein of activity and/or proteolytic enzyme and nuclease, the exchange of DNA chain and the recombinant protein that particularly have proteolytic enzyme and nuclease, particularly recombinant protein recA superfamily is (for example as Table II A or IIB the 5th or 7 row, shown in the 282nd row) activity situation about being enhanced under, preferably, give each fine chemicals in one embodiment, preferably beta-carotene be increased in 43% and 76% or more between.

In one embodiment, at e. coli k12 protein b3172 or its homologue, for example under the situation that the activity of arginyl succsinic acid synthetic enzyme (for example shown in Table II A or IIB the 5th or 7 row, the 283rd row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 35% and 74% or more between.

In one embodiment, at e. coli k12 protein b4129 or its homologue, under the situation that the activity of for example derivable Methionin tRNA synthetic enzyme (for example shown in Table II A or IIB the 5th or 7 row, the 284th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 34% and 63% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, for example under the situation that the activity of NHC protein (for example shown in Table II A or IIB the 5th or 7 row, the 285th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferably beta-carotene be increased in 52% and 108% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR316W or its homologue, for example under the situation that the activity of Jia Ding seven β-thigh family S-adenosylmethionine dependency methyltransgerase (for example shown in Table II A or IIB the 5th or 7 row, the 286th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferably beta-carotene be increased in 28% and 52% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR407C or its homologue, for example participate under the situation that the activity of target complex body (TRAPP) component of ER-Golgi membrane transhipment or its homologue (for example shown in Table II A or IIB the 5th or 7 row, the 287th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 54% and 214% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR513W or its homologue, for example under the situation that the activity of glutathione reductase (for example shown in Table II A or IIB the 5th or 7 row, the 288th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferably beta-carotene be increased in 39% and 43% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YLL013C or its homologue, for example under the situation that PUF protein families (it is from the member pumilio that found and Fbf name) member's (for example shown in Table II A or IIB the 5th or 7 row, the 289th row) activity is enhanced, preferably, give in one embodiment each fine chemicals, preferably beta-carotene be increased in 43% and 50% or more between.

In one embodiment, at e. coli k12 protein b0481 or its homologue, for example have under the situation that the activity of the protein (for example shown in Table II A or IIB the 5th or 7 row, the 637th row) in YbaK-spline structure territory is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 35% and 39% or more between.

In one embodiment, at e. coli k12 protein b0970 or its homologue, for example under the situation that the activity of glutamate receptor (for example shown in Table II A or IIB the 5th or 7 row, the 638th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 36% and 117% or more between.

In one embodiment, at e. coli k12 protein b1736 or its homologue, for example under the situation that the activity of PEP dependency phosphotransferase (for example shown in Table II A or IIB the 5th or 7 row, the 639th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 51% and 137% or more between.

In one embodiment, at e. coli k12 protein b1738 or its homologue, for example under the situation that the activity of PEP dependency phosphotransferase (for example shown in Table II A or IIB the 5th or 7 row, the 640th row) is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 63% and 160% or more between.

In one embodiment, at e. coli k12 protein b3160 or its homologue, for example have under the situation that the activity of the monooxygenase (for example shown in Table II A or IIB the 5th or 7 row, the 641st row) of luciferase sample atpase activity is enhanced, preferably, give in one embodiment each fine chemicals, preferred IPP be increased in 35% and 47% or more between.

[0046.0.24.24] in one embodiment, if e. coli k12 protein b0730 or its homologue, for example succinic thiokinase operon transcriptional and acyl are replied regulatory gene (for example as Table II A or IIB the 5th or 7 row, shown in the 278th row), perhaps b1829 or its homologue, the heat shock protein that for example has protease activity is (for example as Table II A or IIB the 5th or 7 row, shown in the 279th row), perhaps b1926 or its homologue, for example flagellin matter fliT is (for example as Table II A or IIB the 5th or 7 row, shown in the 280th row), perhaps b2211 or its homologue, for example ATP-binding cassette superfamily ATP-binding transport albumen is (for example as Table II A or IIB the 5th or 7 row, shown in the 281st row), perhaps b2699 or its homologue, the exchange of DNA chain and the recombinant protein that for example have proteolytic enzyme and nuclease (for example are listed as Table II A or IIB the 5th or 7, shown in the 282nd row), perhaps b3172 or its homologue, for example arginyl succsinic acid synthetic enzyme is (for example as Table II A or IIB the 5th or 7 row, shown in the 283rd row), perhaps b4129 or its homologue, for example derivable Methionin tRNA synthetic enzyme is (for example as Table II A or IIB the 5th or 7 row, shown in the 284th row) activity situation about being enhanced under; Preferably give each fine chemicals and more multiclass carotene, preferred carotene increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR089C-A or its homologue, for example NHC protein is (for example as Table II A or IIB the 5th or 7 row, shown in the 285th row), perhaps YDR316W or its homologue, for example Jia Ding seven β-thigh family S-adenosylmethionine dependency methyltransgerase is (for example as Table II A or IIB the 5th or 7 row, shown in the 286th row), perhaps YDR407C or its homologue, target complex body (TRAPP) component that for example participates in the transhipment of ER-Golgi membrane is (for example as Table II A or IIB the 5th or 7 row, shown in the 287th row), perhaps YDR513W or its homologue, for example glutathione reductase is (for example as Table II A or IIB the 5th or 7 row, shown in the 288th row), perhaps YLL013C or its homologue, for example PUF protein families (its member pumilio and Fbf name from being found) member is (for example as Table II A or IIB the 5th or 7 row, shown in the 289th row) (for example as Table II A or IIB the 5th or 7 row, shown in the 637th row), perhaps b0481 or its homologue, the protein that for example has YbaK-spline structure territory is (for example as Table II A or IIB the 5th or 7 row, shown in the 638th row), perhaps b0970 or its homologue, for example glutamate receptor is (for example as Table II A or IIB the 5th or 7 row, shown in the 639th row), perhaps b1736 or its homologue, for example PEP dependency phosphotransferase is (for example as Table II A or IIB the 5th or 7 row, shown in the 640th row), perhaps b1738 or its homologue, for example PEP dependency phosphotransferase is (for example as Table II A or IIB the 5th or 7 row, shown in the 641st row), perhaps b3160 or its homologue, for example have under the situation that the activity of the monooxygenase of luciferase sample atpase activity is enhanced, preferably give each fine chemicals and multiclass carotene more, preferred carotene increases.

[0047.0.0.24] to [0048.0.0.24]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.24.24] has to give and improves each fine chemicals quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 5th or 7 row, the 279th, 282,285,286, the sequence of consensus sequence shown in 288 and/or 289 row or as Table I A or IB the 5th or 7 be listed as, the 279th, 282,285,286, its function homologue described in polypeptide or the literary composition shown in 288 and/or 289 row, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 279th, 282,285,286, nucleic acid molecule or its function homologue as herein described shown in 288 and/or 289 row) sequence of coded polypeptide, and have the activity that the β-Hu Luobusu level of giving described in the literary composition increases.

Have to give and improve each fine chemicals quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise as Table IV the 5th or 7 row, the 278th, 280,281,283,284 and/or 287 and/or 637-641 capable shown in consensus sequence sequence or as Table II A or IIB the 5th or 7 row, the 278th, 280,281,283,284 and/or 287 and/or 637-641 capable shown in its function homologue described in polypeptide or the literary composition, perhaps (for example be listed as by Table I A or IB the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, the 278th, 280,281,283,284 and/or 287 and/or 637-641 capable shown in nucleic acid molecule or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the IPP level of giving described in the literary composition increases.

[0050.0.24.24] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, for example, the sylvite of β-Hu Luobusu or IPP or sodium salt, perhaps their ester or glucosides (for example bioside).

[0051.0.24.24] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, can produce the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical), for example comprise the composition of IPP or β-Hu Luobusu.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of IPP or β-Hu Luobusu.

[0052.0.0.0] seen in disclosing of [0052.0.0.24] these paragraphs

[0053.0.24.24] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention (for example has as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 and/or 637-641 capable shown in protein or its homologue (for example Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 and/or 637-641 capable shown in) active polypeptide) express to increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA gives the coded protein of nucleic acid molecule of the present invention (for example to have as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, and/or the 278th, 280,281,283, shown in 284 and/or 287 row) active polypeptide) express to improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or the of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, shown in 284 and/or 287 row) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by the coded protein of nucleic acid molecule of the present invention, polypeptide perhaps of the present invention (for example has as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, and/or the 278th, 280,281,283, shown in 284 and/or 287 row) active polypeptide) express and improve;

(e) by in biological or its part, adding the activity that one or more external source inducible factors come stimulating protein, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, and/or the 278th, 280,281,283, shown in 284 and/or 287 row) active polypeptide) express and improve;

(f) transgenosis of expression coded protein, described protein is given the activity with each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, and/or the 278th, 280,281,283, shown in 284 and/or 287 row) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, described nucleic acid molecule encoding has the active of each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, and/or the 278th, 280,281,283, shown in 284 and/or 287 row) active polypeptide);

(h) by adding positive Expression element or removing negative Expression element and improve code book invention polypeptide and (for example have as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641, the and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row, the 279th, 282,285,286,288 and/or 289 row and/or 637-641 and/or 278,280,281,283, shown in 284 and/or 287 row) active polypeptide) the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or from control region removal straining element.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that each fine chemicals of enhanced produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.24.24] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, (for example Table II A or IIB the 5th or 7 are listed as for protein or its homologue shown in 284 and/or 287 row and/or 637-641 are capable, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide shown in 284 and/or 287 row and/or 637-641 are capable) giving each fine chemicals after active expression of polypeptides or the activity increases.

[0055.0.0.24] to [0064.0.0.24]: see [0055.0.0.0] to [0064.0.0.0] [0065.0.0.24]: see [0065.0.0.0]

[0066.0.0.24] to [0067.0.0.24]: see that [0066.0.0.0] is to [0067.0.0.0]

[0068.0.24.24] be not can cause the mode of disadvantageous effect to introduce sudden change to the generation of each fine chemicals, particularly β-Hu Luobusu or IPP.

[0069.0.0.0] seen in disclosing of [0069.0.0.24] these paragraphs

[0070.0.24.24] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example favourable carotenoid, carotene particularly, particularly the composition of β-Hu Luobusu for example contains high level (from the physiology of nutrition angle) carotenoid, carotene particularly, the composition of β-Hu Luobusu or precursor such as IPP particularly.

[0071.0.0.0] seen in disclosing of [0071.0.0.24] these paragraphs

[0072.0.24.24] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.The example of this compounds is a more carotenoid well-known in the art, for example carotene or carotene, for example ketocarotinoids or hydrogen carotenoid, for example xenthophylls, Lyeopene, alpha-carotene or β-Hu Luobusu or IPP are other compound of its precursor.

[0073.0.24.24] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve the active of polypeptide of the present invention or its homologue (for example Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in) or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation is reclaimed (randomly separating) by free and/or each fine chemicals of bonded of biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis and optional more free and/or bonded carotenoid, particularly carotene, particularly β-Hu Luobusu or its precursor I PP.

[0074.0.24.24] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded, can also produce, reclaim and separate (if desired) other free and/or bonded carotenoid, particularly carotene, particularly β-Hu Luobusu or carotene precursor I PP.

[0075.0.0.24] to [0077.0.0.24]: see that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.24.24] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or be used for other purposes, for example according to following disclosing:

6,399,115：Method?and?composition?for?the?treatment?of?benign?prostatehypertrophy(BPH)and?prevention?of?prostate?cancer；

6,399,114：Nutritional?system?for?nervous?system?disorders；

6,399,060：Composition?having?nematicidal?activity；

6,399,046：Use?of?a?content?of?catechins?or?a?content?of?green?tea?extract?incosmetic?preparations?for?tanning?the?skin；

6,395,782：Method?of?increasing?longevity?and?preventing?body?weightwasting?in?autoimmune?disease?by?using?conjugated?linoleic?acid；

6,395,508：Peptide?mixture?and?products?thereof；

6,395,315：Fermentation?composition，process?for?preparing?the?same，anduse?thereof；

6,395,311：Multicomponent?biological?vehicle；

6,394,230：Sterol?esters?as?food?additives；

6,391,640：Methods?and?compositions?for?cellular?and?metabolicengineering；

6,391,332：Therapeutic?micronutrient?composition?for?severe?trauma，burns?and?critical?illness；

6,391,321：Emulsifier-free?finely?disperse?systems?of?the?oil-in-water?andwater-in-oil?type；

6,391,319：Cosmetic?and?dermatological?emulsions?comprising?alkylglucosides?and?increased?electrolyte?concentrations；

6,391,289：Use?of?sunscreen?combinations?comprising，as?essentialconstituent，4，4′-diarylbutadienes?as?photostable?UV?filters?in?cosmetic?andpharmaceutical?preparations；

6,387,961：Alkyl?2-acetamido-2-deoxyglucopyranoside?and?methods?ofemulsifying；

6,387,927：Epothilone?derivatives?and?their?use?as?antitumor?agents；

6,387,883：Method?for?the?prevention?and?treatment?of?cachexia?andanorexia；

6,387,878：Methods?of?treating?intestinal?ischemia?using?heparin-bindingepidermal?growth?factor；

6,387,862：Bleach?compositions；

6,387,418：Pomegranate?extracts?and?methods?of?using?thereof；

6,387,370：Compositions?containing?extracts?of?Morinda?citrifolia，red?wine，prune，blueberry，pomegranate，apple?and?enzyme?mixture；

6,387,355：Use?of?sunscreen?combinations?comprising，as?essentialconstituent，amino-substituted?hydroxybenzophenones?as?photostable?UVfilters?in?cosmetic?and?pharmaceutical?preparations；

6,384,090：Preparation?of?active?ingredient?dispersions?and?apparatustherefor；

6,384,085：Material?separated?from?Ecklonia?cava，method?for?extractingand?purifying?the?same，and?use?thereof?as?antioxidants；

6,383,751：Assessing?lipid?metabolism；

6,383,543：Process?for?the?extraction?of?an?organic?salt?from?plants，the?salt，and?other?similar?salts；

6,383,524：Compositions?and?methods?for?enhancing?therapeutic?effects；

6,383,523：Pharmaceutical?compositions?and?methods?for?managing?skinconditions；

6,383,503：PREPARATIONS?OF?THE?W/O?EMULSION?TYPE?WITHANINCREASED?WATER?CONTENT，ADDITIONALLY?COMPRISINGONE?OR?MORE?ALKYLMETHICONE?COPOLYOLS?AND/ORALKYLDIMETHICONE?COPOLYOLS，AND，IF?DESIRED，CATIONICPOLYMERS；

6,383,474：Carotenoid?preparation；

6,383,473：Solid?composition?for?reducing?tooth?erosion；

6,380,442：Process?for?the?isolation?of?mixed?carotenoids?from?plants；

6,380,232：Benzimidazole?urea?derivatives，and?pharmaceuticalcompositions?and?unit?dosages?thereof；

6,380,227：Fermentative?preparation?process?for?and?crystal?forms?ofcytostatics；

6,379,697：Stabilization?of?photosensitive?materials；

6,379,683：Nanocapsules?based?on?dendritic?polymers；

6,376,722：Lutein?to?zeaxanthin?isomerization?process?and?product；

6,376,717：Preparation?of?astaxanthin；

6,376,544：Nutritional?product?for?a?person?having?renal?failure；

6,376,498：Pharmaceutical，cosmetic?and/or?food?composition?withantioxidant?properties；

6,376,455：Quaternary?ammonium?compounds，compositions?containingthem，and?uses?thereof；

6,376,005：Antimicrobial?composition?for?food?and?beverage?products；

6,375,993：Pomegranate?extracts?and?methods?of?using?thereof；

6,375,992：Methods?of?hydrating?mammalian?skin?comprising?oraladministration?of?a?defined?composition；

6,375,963：Bioadhesive?hot-melt?extruded?film?for?topical?and?mucosaladhesion?applications?and?drug?delivery?and?process?for?preparationthereof；

6,375,956：Strip?pack；

6,375,873：Process?and?apparatus?for?producing?stably?fine-particlepowders；

6,372,964：For?higher?basidiomycetes?mushrooms?grown?as?biomass?insubmerged?culture；

6,372,946：Preparation?of?4，4′-diketo-.beta.-carotene?derivatives。

The document description of being quoted some embodiment preferred.Some favourable nonrestrictive embodiments have been described in described application.

Can as described in the above-mentioned application or as other method well known to those skilled in the art, for example at Methods in Enzymology:Carotenoids, Part A:Chemistry, Separation, Quantitation and Antioxidation, by John N Abelson or Part B, Metabolism, Genetics, described in the and Biochemistry or described hereinafter method come purified fermentation broth, tunning, plant or plant product.For these different methods of setting up gradually, product is the composition that contains IPP and/or β-Hu Luobusu and optional other carotenoid, its also comprise different amounts, advantageously by weight for 0-99%, preferably be lower than 80% by weight, especially preferably be lower than 50% fermented liquid, vegetable particle and cellular constituent by weight.

[0079.0.0.24] to [0084.0.0.24]: see that [0079.0.0.0] is to [0084.0.0.0]

[0084.2.24.24] the present invention has also considered such embodiment, promptly in each fine chemicals that produces, β-Hu Luobusu or other carotenoid precursors compound such as IPP are present in the spending of selected flowering plant as the host (for example, IPP).The present invention has also considered such embodiment, and promptly the flower of host plant lacks β-Hu Luobusu or other carotenoid precursors, for example Vinca.In the one type plant of back, the DNA that is inserted (if lacking other) comprises the gene of coding carotenoid precursors (can biologically be transformed into the compound of β-Hu Luobusu) and ketolase and hydroxylase.

In one embodiment, preferred flowering plant includes but not limited to: Amaryllidaceae (Amaryllidaceae) (allium (Allium), Narcissus (Narcissus)); Apocynaceae (Apocynaceae) (Vinca (Catharanthus)); Composite family (Compositae or Compositae) (Aster (Aster), calendulin, China aster belong to (Callistephus), Cichorium, golden pheasant Chrysanthemum (Coreopsis), dahlia (Dahlia), Chrysanthemum (Dendranthema), variegated Chrysanthemum (Gazania), African daisy genus (Gerbera), Helianthus, Helichrysum (Helichrysum), Lactuca, Rudbeckia (Rudbeckia), Tagetes, youth-and-old-age genus (Zinnia)); Balsaminaceae (impatiens (Impatiens)); Begoniaceae (Begoniaceae) (begonia (Begonia)); Caryophyllaceae (Carnation (Dianthus)); Chenopodiaceae (Beta, spinach belong to (Spinacia)); Curcurbitaceae (Citrullus (Citrullus), Curcurbita genus, Cucumis (Cucumis)); Cruciferae (Alyssum (Alyssum), Btassica, Erysimum (Erysimum), Matthiola incana (Matthiola), Rhaphanus (Raphanus)); Gentianaceae (Gentinaceae) (Eustoma genus); Mang ox seedling section (Pelargonium); Gramineae (Graminae or Poaceae) (Avena, Hordeum (Horedum), Oryza, Panicum, Pennisetum (Pennisetum), annual bluegrass belong to (Poa), saccharum (Saccharum), Secale, jowar genus, Triticum, Zea); Euphorbiaceae (Euphorbia (poinsettia)); Labiatae (Labiatae) (Salvia (Salvia)); Pulse family (Glycine, Lathyrus (Lathyrus), Medicago (Medicago), Phaseolus, Pisum); Liliaceae (lilium (Lilium)); Lobeliaceae section (lobelia (Lobelia)); Malvaceae (Abelmoschus (Abelmoschus), Gossypium, mallow (Malva)); Plumbaginaceae (Plumbaginaceae) (Statice (Limonium)); Hua Shallot section (Polemoniaceae) (perennial pholx belongs to (Phlox)); Primulaceae (Primulaceae) (Cyclamen (Cyclamen)); Ranunculaceae (Aconitum (Aconitum), Anemone (Anemone), aquilegia (Aquilegia), kincup belong to (Caltha), staggerweed (Delphinium), Ranunculus (Ranunculus)); The Rosaceae (rose (Rosa)); Rubiaceae (Pentas genus); Scrophulariaceae (Scrophulariaceae) (fragrant painted bunting belongs to (Angelonia), antirrhinum (Antirrhinum), HUDIECAO belongs to (Torenia)); Solanaceae (Capsicum, tomato genus, Nicotiana, green winter Solanum (Petunia), Solanum); Umbelliferae (apium, Daucus (Daucus), Pastinaca belong to); Verbenaceae (Verbenaceae) (Verbena (Verbena), Lantana (Lantana)); Violaceae (Viola (Viola)).

[0085.0.24.24] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, perhaps

(b) with the genetic regulatory element that effectively is connected as nucleotide sequence or derivatives thereof shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.24] to [0087.0.0.24]: see that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.24.24] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Because the nutritive value that for example is used for the plant of raise poultry depends on above-mentioned carotene, particularly β-Hu Luobusu and as the total number of the xenthophylls in fodder energy source, so this is very important for the plant breeder.In addition, because the antioxidant level of plant milk extract depends on above-mentioned carotene, particularly β-Hu Luobusu and quantity and/or carotenoid quantity as antioxidant, so this production for make-up composition also is important.

[0088.1.0.0] seen in disclosing of [0088.1.0.24] these paragraphs

[0089.0.0.24] to [0090.0.0.24]: see that [0089.0.0.0] is to [0090.0.0.0]

[0091.0.24.24] therefore, the plant component foreign matter content is low as far as possible, and preferably other foreign matter contents are also low as far as possible, and aforementioned carotene, the particularly β-Hu Luobusu, the particularly fine chemicals that obtain are pure form as far as possible.In these are used, the content of plant component favourable for by weight less than 10%, preferred 1%, more preferably 0.1%, very particularly preferably 0.01% or lower.

[0092.0.0.24] to [0094.0.0.24]: see that [0092.0.0.0] is to [0094.0.0.0]

[0095.0.24.24] advantageously, the pond that increases carotenoid, particularly carotene, particularly β-Hu Luobusu by method of the present invention in genetically modified organism is to separate a large amount of each pure fine chemicals.

[0096.0.24.24] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as the pond of required fine chemicals such as zeaxanthin or cryptoxanthin in the biology) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.Have report, the inhibition of Lyeopene (Lyopene) generation has been increased the quantity of other carotene, particularly β-Hu Luobusu in the cell.In addition, use zeaxanthin or cryptoxanthin can also increase the quantity of chemical described in the cell as the substrate inhibitory enzyme.For example, in one embodiment, if wish to produce the cryptoxanthin or the zeaxanthin of high quantity, the generation that then suppresses astaxanthin is favourable.

Other modification of the glucosides of [0097.0.24.24] zeaxanthin and β-cryptoxanthin, particularly bioside and zeaxanthin and cryptoxanthin is well known by persons skilled in the art.The content that increases each fine chemicals of bonded (for example to the modification of zeaxanthin and cryptoxanthin (particularly its glucosides, as bioside)) also is favourable.

[0098.0.24.24] in preferred embodiments, each fine chemicals be produce according to the present invention and carry out isolating where necessary.The method according to this invention produces other carotenoid, for example carotene or carotene, particularly β-Hu Luobusu, particularly ketocarotinoids or hydrogen carotenoid, for example xenthophylls, Lyeopene, alpha-carotene or β-Hu Luobusu, perhaps each fine chemicals is the compound (for example astaxanthin) of its biosynthesizing precursor compound, perhaps its mixture or with the mixture of other carotenoid (particularly carotene, particularly β-Hu Luobusu) be favourable.

For microbial fermentation, aforementioned purpose fine chemicals can be accumulated in substratum and/or the cell [0099.0.24.24].If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate the composition that comprises each fine chemicals.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.24.24] comprises the method according to this invention synthetic and comprises carotenoid, carotene as described, particularly the transgenic plant of β-Hu Luobusu such as cryptoxanthin or zeaxanthin (perhaps astaxanthin (because it is synthetic from cryptoxanthin or zeaxanthin)) can advantageously directly be put on market, and do not need institute's synthetic carotenoid is separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet, each fine chemicals that the method according to this invention produces can also with the form of its oils, fat, lipid as the extract extract of ether, alcohol or other organic solvent or water (as contain) and/or free carotene, particularly β-Hu Luobusu from biology, advantageously from plant, separate.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by processing or extract realization to plant part.In order to improve the efficient of extraction, vegetable material is cleaned, softens (temper) and shells in case of necessity and peel off is favourable.For example, contain carotene, particularly β-Hu Luobusu oils, fat and/or lipid can need not heat cold be beaten or cold-press process obtains by being called.In order more like a cork plant part (particularly plant seed) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will carry out pretreated seed in this mode squeezes or extracts with solvent (as warm hexane).The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed, promptly come unstuck and/or refining.In this process, at first remove some materials, as plant mucus and suspended matter.By add acid (for example phosphoric acid) can zymetology or or chemistry-physics on influence desliming.

Therefore because carotenoid is positioned at cell in the microorganism, must relate to separation to their recovery to biomass.The method that is used for the good foundation of harvested cell comprises filtration as described herein, centrifugal and condense/flocculate.Must remove the residual carbon hydrogen compound that is adsorbed onto on the cell.Handle for this purpose suggestion use solvent extraction or with tensio-active agent.Yet it is favourable avoiding this processing, because should processing can cause cell to lose most of carotenoid.

[0101.0.24.24] can determine the feature and the purity of institute's separating compound by state of the art.They comprise high performance liquid chromatography (HPLC), gas-chromatography (GC), light-splitting method, mass spectrum (MS), staining, tlc, NIRS, enzymatic determination or microbioassay.These analytical procedures are compiled in: Patek etc. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova etc. (1996) Biotekhnologiya 1127-32; With (1998) Bioprocess Engineer.19:67-70. " Ulmann ' s Encyclopedia of IndustrialChemistry " (1996) Bd.A27 such as Schmidt, VCH Weinheim, the 89-90 page or leaf, the 521-540 page or leaf, the 540-547 page or leaf, the 559-566 page or leaf, 575-581 page or leaf and 581-587 page or leaf; Michal, G (1999) " Biochemical Pathways:An Atlas of Biochemistry and MolecularBiology ", John Wiley and Sons; Fallon, A. etc. (1987) " Applications ofHPLC in Biochemistry in:Laboratory Techniques in Biochemistry andMolecular Biology ", the 17th volume.

[0102.0.24.24] for example, carotene, particularly β-Hu Luobusu, particularly β-cryptoxanthin or zeaxanthin can advantageously detect by HPLC, LC or GC separation method.By using standard method of analysis (LC, LC-MS, MS or TLC) can clearly detect the existence of the product that contains carotene, particularly β-Hu Luobusu, particularly β-cryptoxanthin or zeaxanthin to the biology analysis of recombinating.Can or pass through other applicable method by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding, boiling with material fragmentation to be analyzed.

[0103.0.24.24] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as the sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as the sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, it comprises by use and has the nucleic acid molecule that the primer as sequence shown in Table III the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable obtains amplifier nucleic acid molecule from cDNA library or genomic library, and gives the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as the consensus sequence of sequence shown in Table IV the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, the structural domain of polypeptide shown in described polypeptid acid sequence coding IIA or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[0104.0.24.24] in one embodiment, nucleic acid molecule of the present invention or process of the present invention or method nucleic acid molecule used therefor and Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 the row and/or the 278th, 280,281,283,284 and/or 287 the row and/or 637-641 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention not by Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in polypeptide of sequence.

[0105.0.0.24] to [0107.0.0.24]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.24.24] method of the present invention and had Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the nucleic acid molecule of sequence shown in 284 and/or 287 row and/or 637-641 are capable, from Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, aminoacid sequence shown in 284 and/or 287 row and/or 637-641 are capable is derived or is listed as from containing Table IV the 7th, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 284 and/or 287 row and/or 637-641 are capable, perhaps its coding has as Table II A or IIB the 3rd, 5 or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the enzymic activity of polypeptide or bioactive polypeptide shown in 284 and/or 287 row and/or 637-641 are capable or for example to give each fine chemicals behind its expression or active the increasing be carotenoid, carotene particularly, the derivative or the homologue that increase of β-Hu Luobusu or carotene precursor I PP particularly.

[0109.0.24.24] clones described sequence alone or in combination and to enter in the nucleic acid construct in one embodiment.These nucleic acid constructs make each fine chemicals, particularly IPP or β-Hu Luobusu that the inventive method produced be optimized synthetic.

[0110.0.0.24] sees [0110.0.0.0]

[0111.0.0.0] seen in disclosing of [0111.0.0.24] these paragraphs

The nucleic acid molecule that uses in [0112.0.24.24] the inventive method is the isolated nucleic acid sequences form, its coding has as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide of polypeptide active shown in 284 and/or 287 row and/or 637-641 are capable or have as Table II A or IIB the 5th and 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide of peptide sequence shown in 284 and/or 287 row and/or 637-641 are capable, and give IPP and/or the increase of β-Hu Luobusu level.

[0113.0.0.24] to [0114.0.0.24]: see that [0113.0.0.0] is to [0114.0.0.0]

[0115.0.0.0] seen in disclosing of [0115.0.0.24] these paragraphs

Disclosing of [0116.0.0.24] to [0120.0.0.24] these paragraphs sees that [0116.0.0.0] is to [0120.0.0.0]

[0120.1.0.24] advantageously, the production bacterial strain of selecting in the inventive method is to be selected from following microorganism: green alga such as Spongioccoccum exentricum, Chlorella sorokiniana (Chlorella pyrenoidesa (pyrenoidosa) 7-11-05), perhaps fungi is as belonging to the fungi of colored otology (Daccrymycetaceae), perhaps non-photosynthetic bacterium such as methylotrophy bacterium, Flavobacterium, actinomycetes such as Streptomyces chrestomyceticus, mycobacterium such as Mycobacterium phlei (Mycobacterimphlei) or the red bacterium of pod membrane (Rhodobacter capsulatus).

[0121.0.24.24] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, peptide sequence or its functional homologue as herein described had the difference of one or more amino acid moleculars shown in 284 and/or 287 row and/or 637-641 were capable, described artificial sequence is preferably given aforementioned activity, promptly improving Table II A or IIB the 5th or 7 row, the 279th, 282,285,286, giving the β-Hu Luobusu level after the activity of peptide sequence shown in 288 and/or 289 row increases, and is perhaps improving Table II A or IIB the 5th or 7 row, the 278th, 280,281,283, giving the IPP level after the activity of peptide sequence shown in 284 and/or 287 row and/or 637-641 are capable increases.

[0122.0.0.24] to [0127.0.0.24]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.24.24] polymerase chain reaction (PCR) amplification is used is (for example as Table III the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or the primer of 637-641 shown in capable to) can be based on sequence shown in this paper, for example Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence shown in 284 and/or 287 row and/or 637-641 are capable or from as Table II A or IIB the 5th or 7 be listed as, respective the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence deutero-sequence produced shown in 284 and/or 287 row and/or 637-641 were capable.

[0129.0.24.24] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).Point out in the comparison shown in the figure of the conservative region of polypeptide of the present invention.Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable of described comparison.

[0130.0.24.24] can use then degenerated primer by pcr amplification have aforementioned activity (as improve to express or activity after give each fine chemicals and increase) the new protein fragment or comprise as described in segmental protein.

[0131.0.0.24] to [0138.0.0.24]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.24.24] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with for the Table I A of β-Hu Luobusu or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row or for the Table I A of IPP or IB the 5th or 7 row, the 278th, 280,281,283, sequence hybridization shown in 284 and/or 287 row and/or 637-641 are capable, and the coding expression has each fine chemicals, the i.e. active peptide of β-Hu Luobusu or IPP of increasing.

[0140.0.0.24] to [0146.0.0.24]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.24.24] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.24.24] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, nucleotide sequence or its portion homologous were at least about 30% shown in 284 and/or 287 row and/or 637-641 were capable, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly have the activity that after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue, increases IPP or β-Hu Luobusu.

[0149.0.24.24] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, one of nucleotide sequence shown in 284 and/or 287 row and/or 637-641 are capable or its part hybridization, preferably in as literary composition, hybridize under the defined stringent condition, and coding have above-mentioned activity (as give IPP or β-Hu Luobusu increase) and randomly as Table II A or IIB the 5th be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the protein of protein active shown in 284 and/or 287 row and/or 637-641 are capable.

[00149.1.24.24] randomly, in one embodiment, with Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the nucleotide sequence of one of nucleotide sequence hybridization shown in 284 and/or 287 row and/or 637-641 are capable has another or various active, described activity for as Table II A or IIB the 3rd be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, protein shown in 284 and/or 287 row and/or 637-641 are capable is known activity or is used for these protein of note.

[0150.0.24.24] in addition, nucleic acid molecule of the present invention can only contain Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the part of coding region of one of nucleotide sequence, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give β-Hu Luobusu or IPP increase.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and described nucleotides sequence column region is listed as with Table I A or IB the 5th or 7 under stringent condition, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the sense strand of one of sequence shown in 284 and/or 287 row and/or 637-641 are capable, Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the antisense sequences of one of sequence shown in 284 and/or 287 row and/or 637-641 are capable or its be natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotide hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the right PCR of primer will produce as Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 are gone and/or 637-641 capable shown in fragment or its gene product of polynucleotide sequence.

[0151.0.0.24]: see [0151.0.0.0]

[0152.0.24.24] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and are listed as Table II A or IIB the 5th or 7, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the abundant homology of aminoacid sequence shown in 284 and/or 287 row, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or raising β-Hu Luobusu the (the 279th as be shown in the examples, 282,285,286,288 and/or 289 go) or IPP the (the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable) activity of level.

[0153.0.24.24] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprises and is listed as Table II A or IIB the 5th or 7, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the amino-acid residue that the aminoacid sequence minimal number was identical or of equal value shown in 284 and/or 287 row and/or 637-641 were capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as protein shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or its part have Table II A for example as herein described or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the activity of polypeptide.

[0154.0.24.24] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the complete amino acid sequence homology was at least about 30% shown in 284 and/or 287 row and/or 637-641 were capable, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% also most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.24] to [0156.0.0.24]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.24.24] the present invention relates in addition owing to the genetic code degeneracy is different from Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, one of nucleotide sequence (with its part) shown in 284 and/or 287 row and/or 637-641 are capable and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide of sequence shown in 284 and/or 287 row and/or 637-641 are capable or as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the nucleic acid molecule of polypeptide or its function homologue shown in 284 and/or 287 row and/or 637-641 are capable.Advantageously, nucleic acid molecule of the present invention comprises or has in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or the consensus sequence of 637-641 shown in capable or as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the aminoacid sequence of polypeptide or its function homologue shown in 284 and/or 287 row and/or 637-641 are capable.Also in another embodiment, nucleic acid molecule encoding full length protein of the present invention, described full length protein with comprise as Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or the consensus sequence of 637-641 shown in capable or as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the basic homology of aminoacid sequence of polypeptide or its function homologue shown in 284 and/or 287 row and/or 637-641 are capable.Yet, in a preferred embodiment, nucleic acid molecule of the present invention does not comprise as the sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.

[0158.0.0.24] to [0160.0.0.24]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.24.24] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it is hybridized with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the nucleic acid molecule of sequence) under stringent condition.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.0] seen in disclosing of [0162.0.0.24] these paragraphs

[0163.0.24.24] preferably, under stringent condition with Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals quantity increase in biology or its part (for example tissue, cell or cell chamber) after using activity of proteins).

[0164.0.0.0] seen in disclosing of [0164.0.0.24] these paragraphs

[0165.0.24.24] for example can produce the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place in the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in sequence).

[0166.0.0.24] to [0167.0.0.24]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.24.24] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide are with different as the contained sequence of sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, but have kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contain with as the aminoacid sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable at least about 50% identical aminoacid sequence, and after improving its activity (for example it is expressed), can participate in increasing the generation of each fine chemicals.Preferably, the protein of this nucleic acid molecule encoding with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence shown in 284 and/or 287 row and/or 637-641 are capable is identical at least about 60%, more preferably with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, one of sequence shown in 284 and/or 287 row and/or 637-641 are capable is identical at least about 70%, even more preferably with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence shown in 284 and/or 287 row and/or 637-641 are capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence shown in 284 and/or 287 row and/or 637-641 are capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.24] to [0172.0.0.24]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.24.24] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:34228 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:34228 sequence before use.

[0174.0.0.24]: see [0174.0.0.0]

[0175.0.24.24] for example, the sequence that has 80% homology at protein level and SEQ ID NO:34229 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:34229 sequence.

[0176.0.24.24] is by replacing, insert or lack from being listed as Table II A or IIB the 5th or 7 according to of the present invention, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the functional equivalent that one of polypeptide obtained shown in 284 and/or 287 row and/or 637-641 were capable be listed as according to of the present invention as Table II A or IIB the 5th or 7, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, one of polypeptide had at least 30% shown in 284 and/or 287 row and/or 637-641 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide shown in 284 and/or 287 row and/or 637-641 are capable has essentially identical character and discerns.

[0177.0.24.24] is by replacing, insert or lack from being listed as Table I A or IB the 5th or 7 according to of the present invention, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the functional equivalent that obtains of nucleotide sequence shown in 284 and/or 287 row and/or 637-641 are capable be listed as according to of the present invention as Table II A or IIB the 5th or 7, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, one of polypeptide had at least 30% shown in 284 and/or 287 row and/or 637-641 were capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and coding have with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide of the essentially identical character of polypeptide shown in 284 and/or 287 row and/or 637-641 are capable.

[0178.0.0.0] seen in disclosing of [0178.0.0.24] these paragraphs

[0179.0.24.24] can (particularly be listed as Table I A or IB the 5th or 7 by the nucleotide sequence to nucleic acid molecule of the present invention, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, shown in 284 and/or 287 row and/or 637-641 are capable) the middle replacement of introducing one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II A or IIB the 5th or 7 and be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the nucleic acid molecule of the homologue of protein sequence shown in 284 and/or 287 row and/or 637-641 are capable.Can pass through standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) and introduce sudden change in the encoding sequence shown in capable as Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641.

[0180.0.0.24] to [0183.0.0.24]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.24.24] is employed to have as Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the nucleotide sequence homologous compound of sequence shown in 284 and/or 287 row and/or 637-641 are capable, perhaps come Table II A or IIB the 5th or 7 row freely, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the homologous compound of nucleotide sequence of sequence shown in 284 and/or 287 row and/or 637-641 are capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.24.24] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprises one or more as the sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.In one embodiment, the preferred nucleic acid molecule comprises other Nucleotide that does not show in the least possible, the arbitrary sequence shown in capable as Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method is with identical as the sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.

The employed one or more nucleic acid molecule encodings of [0186.0.24.24] also preferred the inventive method comprise as polypeptide of sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method is with identical as sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.

[0187.0.24.24] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprises as polypeptide of sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable and contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, employed nucleic acid molecule and coding are identical as the encoding sequence of sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable in the method.

[0188.0.24.24] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II A or IIB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, shown in 284 and/or 287 row and/or 637-641 are capable and under the same conditions the polypeptide expressed activity is compared and basic is not reduced.

[0189.0.24.24] as the homologous compound of sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, or deutero-also refers to the single stranded DNA or the RNA of truncated sequence, cDNA, coding and noncoding DNA sequence as the homologue of the sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.24]: see [0190.0.0.0]

[0191.0.0.24] compares with biological or its part of described contrast or selection in one embodiment, according to the inventive method described herein should biology or its part each fine chemicals level of producing protein bound improve.

[0192.0.0.24] to [0203.0.0.24]: see that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.24.24] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding at least mature form as polypeptide or its segmental nucleic acid molecule shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, described nucleic acid molecule is given each fine chemicals in biological or its part, promptly β-Hu Luobusu (the 279th, 282,285,286,288 and/or 289 row) or IPP (the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable) quantity increase;

(b) comprise, preferably comprise mature form at least as nucleic acid molecule or its segmental nucleic acid molecule shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, described nucleic acid molecule is given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the nucleic acid molecule that primer shown in 284 and/or 287 row and/or 637-641 are capable or primer obtain amplification from cDNA library or genomic library, and in biological or its part, give each fine chemicals, promptly β-Hu Luobusu the (the 279th, 282,285,286,288 and/or 289 go) or IPP the (the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable) increase of quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contain just like Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in consensus sequence and in biological or its part, give each fine chemicals, the i.e. nucleic acid molecule that increases of β-Hu Luobusu (the 279th, 282,285,286,288 and/or 289 row) or IPP (the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable) quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, be the nucleic acid molecule that β-Hu Luobusu (the 279th, 282,285,286,288 and/or 289 row) or IPP (the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable) quantity increase, the structural domain of the polypeptide of described peptide coding shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I A or IB the 5th or 7, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, nucleic acid molecule shown in 284 and/or 287 row and/or 637-641 are capable or coding (optimized encoding is mature form at least) are as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, at least the 15nt of the nucleic acid molecule of polypeptide shown in 284 and/or 287 row and/or 637-641 are capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as the sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention do not form by Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 by the sequence shown in capable.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in sequence at least 30% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as the peptide sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.Therefore, in one embodiment, polypeptide shown in nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable at least one or the different polypeptide of a plurality of amino acid, and therefore do not encode as the protein of sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.Therefore, in one embodiment, therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by forming as sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, protein sequence at least 30% shown in 284 and/or 287 row and/or 637-641 are capable identical and with Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence was less than 100% shown in 284 and/or 287 row and/or 637-641 were capable, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% is identical.

[0205.0.0.24] to [0206.0.0.24]: see that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.24.24] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is gene or its combination of carotenoid metabolism, carotene, particularly β-Hu Luobusu metabolism, astaxanthin metabolism, amino acid metabolism, glycolysis-, Tricarboxylic Acid Metabolism.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.24] to [0226.0.0.24]: see that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.24.24] can clone aforementioned nucleic acid molecule and other assortment of genes in nucleic acid construct into of the present invention or the carrier together, perhaps by transform some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing in host cell (advantageously being vegetable cell or microorganism).

Except Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or the capable sequence or derivatives thereof of mentioning of 637-641, can advantageously in biology, express and/or other genes that suddenly change.Particularly advantageously, extra other genes (for example one of said gene of this approach) or astaxanthin or another provitamin A or another carotenoid or other genes of carotene synthetic of expressing at least one carotenoid, particularly carotene biosynthetic pathway in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required fine chemicals, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.24.24] therefore cultivated in another embodiment of the present invention and crossed expression coding and carotenoid, particularly carotene metabolism (particularly β-cryptoxanthin, zeaxanthin, astaxanthin or xenthophylls are synthetic) at least one nucleic acid of related protein or the biology of a gene simultaneously.

[0229.0.24.24] can make up the sequence of other favourable nucleotide sequences of expression for other gene of coding carotenoid biosynthetic pathway with sequence that present method is used and/or aforementioned biosynthesis gene, for example (it is the important control point of flux adjusting to phytoene synthase (Psy), see Fraser etc., 2002), phytoene desaturase (Pds), z-carotene desaturase, above-mentioned enzyme (seeing the preface part of application) for example hydroxylase such as β-Hu Luobusu hydroxylase (US 6,214,575), (US 6 for ketolase or cyclase such as beta cyclase, 232,530) or oxygenase such as US 6,218, β-C4-oxygenase or its homologue described in 599, (US 6 for the astaxanthin synthase, 365,386), perhaps US 6,150, other gene of describing in 130.These genes cause the synthetic increase of essential carotenoid, particularly carotene, particularly β-Hu Luobusu.

[230.0.0.0] seen in disclosing of [0230.0.0.24] these paragraphs.

[0231.0.24.24] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened degraded β-Hu Luobusu or IPP simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.24] to [0276.0.0.24]: see that [0232.0.0.0] is to [0276.0.0.0]

[0277.0.24.24] can separate each fine chemicals that produces by the method that the technician is familiar with from biology, for example by extract, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.The fine chemicals of producing by this process is that β-Hu Luobusu or IPP and other carotenoid, particularly carotene can be by from crop (biology is grown therein) or the biological acquisitions of field results.This can be by squeezing plant part or extract realization.

[0278.0.0.24] to [0282.0.0.24]: see that [0278.0.0.0] is to [0282.0.0.0]

[0283.0.24.24] in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, protein shown in 284 and/or 287 row and/or 637-641 are capable, perhaps as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the antibody of polypeptide shown in 284 and/or 287 row and/or 637-641 are capable, it can utilize polypeptide of the present invention or its fragment (being polypeptide of the present invention) to produce by standard technique.Monoclonal antibody preferably.

[0284.0.0.0] seen in disclosing of [0284.0.0.24] these paragraphs

[0285.0.24.24] the present invention relates to have as sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or by as the coded polypeptide of sequence of nucleic acid molecule or its function homologue shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable in one embodiment.

[0286.0.24.24] is in an advantageous embodiment, improved in the inventive method and contained just like Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, consensus sequence shown in 284 and/or 287 row and/or 637-641 are capable or by the activity of its polypeptide of forming, and in another embodiment, the present invention relates to contain just like Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, consensus sequence or by its polypeptide of forming shown in 284 and/or 287 row and/or 637-641 are capable, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can perhaps can be substituted and/or lack in another embodiment by arbitrary aminoacid replacement.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to and comprising more than a polypeptide as consensus sequence (each row) shown in Table IV the 7th row, 287-289 and/or 637-641 are capable.

[0287.0.0.24] to [0289.0.0.24]: see that [0287.0.0.0] is to [0289.0.0.0]

[00290.0.24.24] uses Vector NTI Suite 8.0, (InforMax ^TM, Invitrogen ^TMLifescience software, U.S.Main Office, 7305Executive Way, Frederick, MD21704, U.S.) assembly AlignX software (on September 25th, 2002) and use following setting to compare: for the pairing comparison: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Right for multiple ratio: breach is opened point penalty: 10.0; Breach extends point penalty 0.1; Breach separates the point penalty scope: 8; Residue is replaced matrix: blosum62; Hydrophilic residue: G P S N D Q E K R; Conversion weight: 0.5; Total calculation options: total residue mark: 0.9.Also selected to set in advance in order to compare conserved amino acid.

[0291.0.24.24] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as the sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable by one or more amino acid.In one embodiment, polypeptide passes through more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence shown in 284 and/or 287 row and/or 637-641 are capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence shown in 284 and/or 287 row and/or 637-641 are capable.In another embodiment, described polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.

[0292.0.0.0] seen in disclosing of [0292.0.0.24] these paragraphs

[0293.0.24.24] the present invention relates to give that fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention in one embodiment in biological or its part.In one embodiment, polypeptide of the present invention have by one or more amino acid with as other sequence of sequence phase region shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.In another embodiment, polypeptide of the present invention can't help to form as sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide not by Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.24.24] in one embodiment, the present invention relates to have as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide of activity of proteins shown in 284 and/or 287 row and/or 637-641 are capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, sequence shown in 284 and/or 287 row and/or 637-641 are capable.

[0295.0.0.24] to [0296.0.0.24]: see that [0295.0.0.0] is to [0296.0.0.0]

[0297.0.0.0] seen in disclosing of [0297.0.0.24] these paragraphs

The chemical of [00297.1.24.24] non-polypeptide of the present invention be for example do not have Table II A or IIB the 3rd, 5 or 7 row, 278-289 is capable and/or 637-641 capable shown in the active polypeptide of polypeptide.

[0298.0.24.24] polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as the abundant homologous aminoacid sequence of aminoacid sequence shown in capable with Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as the identical aminoacid sequence of sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.

[0299.0.24.24] in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I A or IB the 5th or 7 be listed as, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the homology of one of nucleotide sequence sequence shown in 284 and/or 287 row and/or 637-641 are capable is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprises and nucleotide sequence or the coded aminoacid sequence of its homologue as nucleotide sequence hybridization (preferred hybridize under stringent condition) shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable.

[0300.0.24.24] therefore, described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with different as sequence shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 capable and/or 637-641 are capable.Therefore, this polypeptide contains and is listed as Table II A or IIB the 5th or 7, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the complete amino acid sequence homology of sequence was at least about 35% shown in 284 and/or 287 row and/or 637-641 were capable, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.24]see[0301.0.0.0]

The biologically-active moiety of [0302.0.24.24] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the aminoacid sequence shown in 284 and/or 287 row and/or 637-641 are capable or the aminoacid sequence of its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.0] seen in disclosing of [0303.0.0.24] these paragraphs

[0304.0.24.24] operation nucleic acid molecule of the present invention may cause generation have basically as the active of polypeptide shown in Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.24] to [0308.0.0.24]: see that [0305.0.0.0] is to [0308.0.0.0]

[0309.0.0.24] in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, protein shown in 284 and/or 287 row and/or 637-641 are capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, and be not shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide that " non-polypeptide of the present invention " or " other polypeptide " during 284 and/or 287 row and/or 637-641 are capable is meant the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide shown in 284 and/or 287 row and/or 637-641 are capable is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are proteinic and from the protein of identical or different biology shown in capable.In one embodiment, be not shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or capable " the non-polypeptide of the present invention " or " other polypeptide " of 637-641 and do not give each fine chemicals increase in biology or its part.

[0310.0.0.24] to [0334.0.0.24]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.24.24] confirmed that the dsRNAi method is to reducing as the expression of the nucleotide sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reducing the double stranded rna molecule (dsRNA molecule) that causes the metabolic activity change as the expression of nucleotide sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable and/or its homologue.At the double stranded rna molecule that is used for reducing as the coded protein expression of the nucleotide sequence of sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.24] to [0342.0.0.24]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.24.24] is as describing, in order to cause effective reduction of expression, dsRNA and as Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example, can be used for suppressing expressing accordingly in another organism from the dsRNA that begins to produce as sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or its homologue in a kind of organism.

[0344.0.0.24] to [0350.0.0.24]: see that [0344.0.0.0] is to [0350.0.0.0]

[0351.0.0.24] to [0361.0.0.24]: see that [0351.0.0.0] is to [0361.0.0.0]

[0362.0.24.24] therefore, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide shown in 284 and/or 287 row and/or 637-641 are capable, for example coding has protein such as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the polypeptide of polypeptide active shown in 284 and/or 287 row and/or 637-641 are capable) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Have as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the transgenosis of the polypeptide of polypeptide active was meant in the text because genomic regulation and control or manipulation shown in 284 and/or 287 row and/or 637-641 were capable, in cell or biological or its part, be noted as Table II A or IIB the 3rd row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide shown in 284 and/or 287 row and/or 637-641 are capable (for example has as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, polypeptide of sequence shown in 284 and/or 287 row and/or 637-641 are capable) activity is enhanced.Example and the inventive method are described in above.

[0363.0.0.0] seen in disclosing of [0363.0.0.24] these paragraphs

[0364.0.24.24] is when by non-natural synthetic " manually " method (as mutagenesis) when modifying, naturally occurring expression cassette---for example naturally occurring coding such as Table II A or IIB the 3rd row, 5 or 7, the 279,282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the promotor of the gene of polypeptide of the present invention shown in 284 and/or 287 row and/or 637-641 are capable is listed as Table I A or IB the 5th or 7 with corresponding, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the combination of protein coding sequence shown in 284 and/or 287 row and/or 637-641 are capable---become transgene expression cassette.These methods be described (US 5,565,350; WO 00/15815; Be also shown in above).

[0365.0.0.24] to [0373.0.0.24]: see that [0365.0.0.0] is to [0373.0.0.0]

[0374.0.24.24] contains the transgenic plant of synthesizing each fine chemicals in the methods of the invention and can directly introduce to the market, and do not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, each fine chemicals that produces in the inventive method also can be from plant with free β-Hu Luobusu or free IPP or be bonded to compound or the isolated in form of part (for example glucoside, for example diglucoside).Each fine chemicals that produces by this method can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.24] to [0376.0.0.24]: see that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.24.24] therefore the invention still further relates to the method according to this invention, separated the carotenoid that is produced thus, for example comprises the composition of fine chemicals.In one embodiment, separated each fine chemicals that is produced.

[0378.0.24.24] can separate by this way and be higher than 50% by weight, and favourable is higher than 60%, preferably is higher than 70%, especially preferably is higher than 80%, extremely preferably is higher than each fine chemicals that produces in the method for 90%.As required, resultant each fine chemicals can be further purified subsequently, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.24.24] is fine chemicals by the product that the present invention produced in one embodiment.In one embodiment, product is the mixture of each fine chemicals and other carotenoid.

The suitable synthetic parent material of each fine chemicals that [0380.0.24.24] obtains by the inventive method as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises that separation produces each fine chemicals (if expectation) by the composition that comprises each fine chemicals that the inventive method produced or by the inventive method, but and with pharmaceutically acceptable carrier or can be used for the carrier formulated product of makeup or product is mixed with the form of agricultural application.Another embodiment of the present invention be each fine chemicals of producing of the inventive method or genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine purposes or be used to produce the purposes of other carotenoid, for example after separating each fine chemicals, perhaps do not separate but be used for the used biological in-situ of the inventive method that each fine chemicals produces.

[0381.0.0.24] to [0382.0.0.24]: see that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.24.24]./.

[0384.0.0.0] seen in disclosing of [0384.0.0.24] these paragraphs

The fermented liquid that [0385.0.24.24] obtains with this kind approach, particularly contain fermented liquid with other carotenoid, particularly other carotenoid, particularly carotene blended IPP or β-Hu Luobusu, perhaps contain and comprise each fine chemicals or the microorganism of carotenoid or the fermented liquid of microorganism part (as plastid) that is produced with other carotenoid, particularly other carotene blended, usually dry matter content accounts for 1 to 70% of weight, preferred 7.5 to 25%.When finishing, for example passed through fermentation time at least 30% the time to carry out sugared restricted fermentation be especially favourable.This means that the concentration that can utilize sugar in the fermention medium during this period of time remains on or be reduced to 0 to 10g/l, preferred 0 to 3g/l.Then fermented liquid is further handled.As requested, the composite entity that biomass can be by separation method (for example centrifugal, filtration, decant, condense/flocculate) or these methods or partly from fermented liquid, shift out or separate or stay wherein fully.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

Because carotenoid is usually located at film or plastid, therefore in one embodiment, when by separation method such as centrifugal, filtration, decant, condense/flocculate or the combination of these methods from the fermented liquid kind completely or partially during the separating bio amount, avoiding cell is favourable by filtration.The biomass of doing directly can be added in the animal-feed, as long as the enough height and do not have toxic chemical of the concentration of carotenoid.Consider the unstable of carotenoid, drying conditions such as spraying drying or expansion drying are wanted gentle and will be avoided oxidation and suitable/anti-isomerization.Can add antioxidant, for example BHT, ethoxyquinoline or other antioxidant.Under the concentration of carotenoid situation to be diluted, can use solvent extraction to separate, for example use alcohol, ether or other organic solvent (as methyl alcohol, ethanol, acetone, potassium hydroxide alcohol, phenol glycerine, liquefied phenol) or with acid or alkali such as trichoroacetic acid(TCA) or potassium hydroxide.Can find separation carotenoid in current state of the art, particularly the multiple favorable method and the technology of each fine chemicals, particularly IPP or β-Hu Luobusu.Under the situation of using phenol, phenol can remove with ether and water extraction, and the exsiccant elutriant comprises biomass carotenoid mixture.

[0386.0.24.24] therefore can also be further purified each fine chemicals or other carotenoid, the particularly carotene that produce according to the present invention.For this reason, the composition that will contain product, the lipid extracts fraction total or part that with an organic solvent obtains is as mentioned above for example carried out (but being not limited to) saponification (to remove triacylglycerol), distributes (distributing) (from more separating nonpolar epiphase the low phasic property derivative of polar) and separate (by for example open column chromatography or HPLC) between hexane/methanol, and wherein purpose product or impurity are all or part of stays on the chromatography resin.If desired, can use identical or different chromatography resin to repeat these chromatographic steps.The technician is familiar with the effective use of the selection of suitable chromatography resin and they.

[0387.0.0.24] to [0392.0.0.24]: see that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.24.24] the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell in one embodiment, and it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) identify the nucleic acid molecule of particularly under lax stringent condition, hybridizing, and randomly separate full length cDNA clone or complete genome group clone as the nucleic acid molecule shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable with nucleic acid molecule of the present invention;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.24] to [0398.0.0.24]: see that [0394.0.0.0] is to [0398.0.0.0]

[0399.0.24.24] in one embodiment, the present invention relates to identify the method for giving the compound that each fine chemicals generation increases in plant or the microorganism in addition, and it comprises step:

(a) culturing cell or tissue or microorganism or keep plant under proper condition, the nucleic acid molecule of these cell or tissues or microorganism or expression of plants polypeptide of the present invention or coding said polypeptide and can the interactional read-out system of homopolypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and can be provided under the condition of the expression of polypeptides that uses in the described read-out system of permission and polypeptide of the present invention or the inventive method and respond compound and described polypeptide bonded detectable signal; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

Can cultivate biological (as microorganism) under each fine chemicals biosynthesis inhibitor by the reduction amount of growing in existence, thereby the gene product or the agonist of each fine chemicals generation raising are given in screening.Compared with the control, growth preferably as higher mitogenetic rate or high dry matter will identify gene or gene product or the agonist of giving each fine chemicals generation raising under these conditions.

Whether [00399.1.24.24] it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect and depends on as Table II A or IIb the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, active or the expression of the polypeptide shown in 284 and/or 287 row and/or 637-641 are capable or its homologue, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283, the biology phenotype more much at one of activity of proteins shown in 284 and/or 287 row and/or 637-641 are capable.

[0400.0.0.24] to [0415.0.0.24]: see that [0400.0.0.0] is to [0415.0.0.0]

[0416.0.0.0] seen in disclosing of [0416.0.0.24] these paragraphs

[0417.0.24.24] nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce carotenoid, the particularly inhibitor of carotene biosynthetic pathway are had the biology of resistance.Particularly, expressing excessively of polypeptide of the present invention or the used polypeptide of the inventive method may make biology, and for example microorganism or plant are anti-can block carotenoid or carotene (particularly each fine chemicals) synthetic inhibitor in the described biology.

The example of carotenoid synthetic inhibitor or weedicide can be divided into two groups in biological for example microorganism of blocking-up or the plant.First group comprises the early stage intermediate product that causes in this approach, the inhibitor of particularly colourless plant alkene accumulation, for example pentanoic.Other inhibitor is preferably blocked the late phase reaction in this approach, particularly cyclization of lycopene.Inhibitor is nicotine, 2-(4-chlorobenzene sulfo-)-triethylamine and other replacement amine and nitrogen heterocyclic ring base, for example imidazoles.

Biological antioxidant is coerced, the particularly damage of singlet oxygen because carotene can be protected, and the level that therefore increases each fine chemicals can the anti-weedicide that causes producing oxygenated compound (for example singlet oxygen) of protective plant.For example, the inhibition to a kind of important enzyme proporphyrinogen oxidase (Protox) in chlorophyll and the protoheme biosynthesizing causes the loss of Chlorophylls and Carotenoids and causes producing the seepage film; The destruction of film is owing to the generation (weedicide for other typical photosynthetic inhibitor that becomes second nature also has report) of free oxygen free radical.

Therefore, in one embodiment, the increase of each fine chemicals level is used for the anti-weedicide that destroys film (because generation of free oxygen free radical) of protective plant.

Producing the inhibitor of oxidative stress or the example of weedicide is aryl triazoles (aryl triazion), for example sulfentrazone, fluorine ketazolam grass; Perhaps diphenyl ether, for example acifluorfen, lactofen or Oxyfluorfen; Perhaps N-phenylphthalimide, for example imide phenoxy acetic acid Huo Fu oxazinone; Substituted urea class, for example fluometuron, Metribuzin, Diuron Tech or methoxydiuron; Perhaps triazines, for example G-30027, prometryn, ametryn, sencorex, prometon, simazine or six piperazines with; Perhaps uracil, for example bromacil or terbacil.

The carotenoid inhibitor is for example pyridine and pyridazinone, for example monometflurazone, fluorine humulone or dithiopyr.Therefore, in one embodiment, the present invention relates to improve the purposes according to each fine chemicals of the present invention, it is used for the injury that protective plant is avoided carotenoid inhibitor such as pyridine and pyridazinone.

[0418.0.0.24] to [0423.0.0.24]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.24.24] therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other carotenoid, particularly other carotene such as astaxanthin, alpha-carotene or gamma carotene etc.

Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0424.1.0.24] the present invention relates to the purposes that antagonist of the present invention, plant of the present invention or its part, microorganism of the present invention or host cell or its part are used for production cosmetic compositions or pharmaceutical composition in other embodiments.This composition has anti-oxidant activity, the light protection is active, tanning is active, but for example it is used for the treatment of high-level cholesterol and/or fat with pharmaceutically acceptable carrier or cosmetic carrier combinations; Be used for protection, treat or cure above-mentioned disease (for example retinal diseases, hypercholesterolemia, hyperlipidemia and arteriosclerosis); Perhaps be used for cleaning, conditioning and/or handle skin.

Each fine chemicals, particularly β-Hu Luobusu can be used as the stablizer of other color sensitivity or oxygen sensitive compound.

[0425.0.0.24] to [0434.0.0.24]: see that [0425.0.0.0] is to [0434.0.0.0]

[0435.0.24.24] embodiment 3: the interior and vitro mutagenesis of body

[0436.0.24.24] can be by the plasmid DNA (or other carrier DNAs) that will contain one or more purpose nucleotide sequences, nucleic acid molecule for example of the present invention or carrier of the present invention change the intestinal bacteria that can not keep its genetic information integrity or other microorganisms over to (as some kind of bacillus or yeast, as yeast saccharomyces cerevisiae) implement green alga (Spongiococcum sp (for example Spongiococcumexentricum) for example, Chlorella certain (Chlorella sp.)), yeast belong, genus mortierella, the mutagenesis in vivo of Escherichia and above-mentioned other genus, be beneficial to produce carotenoid, for example carotene, particularly β-Hu Luobusu or its precursor I PP.Usually, mutator contains sudden change (as mutHLS, mutD, mutT etc. in the gene of DNA repair system; More visible Rupp, W.D., (1996), and DNA repair mechanisms in Escherichia coli and Salmonella, the 2277-2294 page or leaf, ASM:Washington).Those skilled in the art understand these bacterial strains.The use of these bacterial strains is at for example Greener, A. and Callahan, and M., 1994, have illustrated among the Strategies 7:32-34.

It is that those skilled in the art are well-known that external mutation method for example improves spontaneous mutation rate by chemistry or physical treatment.Mutagenic compound such as 5-bromouracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methane sulfonate (=EMS), azanol and/or nitrous acid is to be widely used in the chemicals of vitro mutagenesis at random.The most frequently used physical mutagenesis method is to use the UV radiation treatment.Another kind of random mutagenesis technology is that amino acid change is imported proteinic fallibility PCR.During PCR, import sudden change wittingly by use fallibility archaeal dna polymerase and special reaction condition well known by persons skilled in the art.For this method, randomized dna sequence dna importing expression vector and resulting sudden change as described below library are used to screen the protein active that changes or improve.

Site-directed mutagenesis method (importing the expection sudden change as using M13 or phagemid carrier and short Oligonucleolide primers) is well-known site-directed mutagenesis method.The key of this method is nucleotide sequence of the present invention is cloned into M13 or phagemid carrier, and M13 or phagemid carrier allow to reclaim the strand recombinant nucleic acid sequence.Design the mutagenic oligonucleotide primer then, its sequence except a single difference fully with the zone that will suddenly change in nucleic acid array complementation: in the expection mutational site its have one with expection suddenly change the Nucleotide complementation and not with initial Nucleotide complementary base.Allow mutagenic oligonucleotide to start the synthetic complementary full length sequence that comprises the expection sudden change with generation of new DNA then.Another kind of site-directed mutagenesis method is the PCR mispairing primer mutafacient system that the technician has also known.The DpnI site-directed mutagenesis is another kind of known method, for example is described in the scheme of Stratagene QuickchangeTM site-directed mutagenesis test kit.A large amount of other methods also are known and are used for general practice.The biology that produces each fine chemicals of expection by screening can screen positive catastrophic event.

[0437.0.24.24] embodiment 4: the DNA between intestinal bacteria, yeast saccharomyces cerevisiae and Mortierella alpina (Mortierellaalpina) shifts

Shuttle vectors such as pYE22m, pPAC-ResQ, pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10, pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101 and its derivative that [0438.0.24.24] allows nucleotide sequence to shift between intestinal bacteria, yeast saccharomyces cerevisiae and/or Mortierella alpina are that the technician is available.The simple method of separating this type of shuttle vectors is by Soni R. and Murray J.A.H.[Nucleic Acid Research, and the 20th rolls up the 21st phase, 1992:5852] open.If desired, this type of shuttle vectors can use and add the replication orgin that is used for intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina or from the standard escherichia coli vector and/or the above-mentioned carrier (Sambrook of the suitable mark of intestinal bacteria, yeast saccharomyces cerevisiae or Mortierella alpina, J. etc., (1989), " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Laboratory Press or Ausubel, F.M. etc. (1994) " CurrentProtocols in Molecular Biology ", John Wiley ﹠amp; Sons) easily make up.These replication orgin are preferably taken from from process of the present invention and are used isolating endogenous plasmid the species.The special gene that uses as the transformation marker of these species is kalamycin resistance (as deriving from Tn5 or Tn-903 transposon) or chlorampenicol resistant (Winnacker, E.L. (1987) " From Genes toClones-Introduction to Gene Technology ", VCH, Weinheim) gene or other antibiotics resistance gene, for example G418, gentamicin, Xin Meisu, Totomycin or kalamycin resistance gene.

[0439.0.24.24] uses standard method goal gene can be cloned in the lump these heterozygosis carriers being introduced in the process bacterial strain uses therefor of the present invention of aforementioned shuttle vectors.The conversion of yeast belong can transform (Bishop etc., Mol.Cell.Biol., 6,1986:3401-3409 by LiCl or spheroplast (sheroplast); Sherman etc., Methods in Yeasts in Genetics, [Cold SpringHarbor Lab.Cold Spring Harbor, N.Y.] 1982, Agatep etc., Technical TipsOnline 1998,1:51:P01525 or Gietz etc., Methods Mol.Cell.Biol.5,1995:255-269) or electroporation (Delorme E., Appl.Environ.Microbiol., the 55th the volume, the 9th phase, 1989:2242-2246) realize.

[0440.0.24.24] for example is integrated into yeast or fungal gene group if the sequence that transforms need advantageously be integrated into the genome of microorganism that process of the present invention is used, also has standard technique known to the skilled for this purpose.(Proc Natl Acad Sci U S A. such as Solinger, 2001 (15): 8447-8453) and (Genetics such as Freedman, the 162nd volume, 15-27, in September, 2002) taught the homologous recombination system that depends on rad 50, rad51, rad54 and rad59 in the yeast.The carrier that use is used for this system of homologous recombination derives from Yip series.For example to derive from the plasmid vector of 2 μ-carrier be well known by persons skilled in the art and be used in the yeast and express.Other preferred carrier is for example pART1, pCHY21 or pEVP11, (EMBO J.1987 by McLeod etc. for they, 6:729-736) and (Genes Dev.5 such as Hoffman, 1991::561-571.) or Russell etc. (J.Biol.Chem.258 1983:143-149.) is described.Other useful yeast vector is REP, REP-X, pYZ or RIP series plasmid.

[0441.0.0.0] seen in disclosing of [0441.0.0.24] these paragraphs

[0442.0.0.0] seen in disclosing of [0442.0.0.24] these paragraphs

[0443.0.0.0] seen in disclosing of [0443.0.0.24] these paragraphs

[0444.0.24.24] embodiment 6: the biology of cultivating genetic modification: substratum and culture condition

[0445.0.24.24] cultivates yeast, genus mortierella or the intestinal bacteria of genetic modification in synthetic or natural medium well known by persons skilled in the art.Being used for culturing yeast, genus mortierella or colibacillary multiple different growth mediums is well-known and can extensively obtains.Cultivating the method for genus mortierella is described by [Bot.Bull.Acad.Sin. (2000) 41:41-48] such as Jang.Genus mortierella can be cultivated down for 6.5,20 ℃ in pH in the substratum that contains 10g/l glucose, 5g/l yeast extract.In addition, Jang etc. has taught and has contained 20g/l Zulkovsky starch, 5g/l bacterium with yeast extract, 10g/l KNO ₃, 1g/l KH ₂PO ₄With 0.5g/l MgSO ₄7H ₂The submergence basic medium of O (pH 6.5).

[0446.0.0.24] to [0450.0.0.24]: see that [0446.0.0.0] is to [0450.0.0.0]

[0451.0.5.5] seen in disclosing of [0451.0.0.24] these paragraphs

[0452.0.0.24] to [0454.0.0.24]: see that [0452.0.0.0] is to [0454.0.0.0]

[0455.0.24.24] can be by cultivating down the microorganism of modifying or modification at felicity condition (as indicated above) plant and analyze substratum and/or the influence that genetic modification produces purpose compound (for example lipid acid) in plant, fungi, algae or the ciliate is measured in raising that the purpose product of cellular component (being lipid or lipid acid) produces.These analytical technologies are known by the technician, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of Industrial Chemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and Molecular Biology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery and purification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recovery processes for biologicalMaterials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques in biotechnology ", Noyes Publications).

Except said process, as (1999) Proc.Natl.Acad.Sci.USA96 (22) such as Cahoon: from vegetable material, extract plant lipid as described in (1986) Analytic Biochemistry152:141-145 such as 12935-12940 and Browse.The qualitative and quantitative analysis of lipid is described in Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:Oily Press (Oily Press Lipid Library; 2); Christie, William W., GasChromatography and Lipids.A Practical Guide-Ayr, Scotland:Oily Press, 1989, Repr.1992, IX, 307pp. (Oily Press Lipid Library; 1); " Progress inLipid Research, Oxford:Pergamon Press, 1 (1952)-16 (1977), exercise question is Progress in the Chemistry of Fats and Other Lipids CODEN.

[0456.0.0.24]: see [0456.0.0.0]

[0457.0.24.24] embodiment 9: the purifying of carotene and assay

[0458.0.24.24] abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By using described standard method of analysis LC, LC-MSMS or TLC can clearly detect the existence of β-Hu Luobusu or IPP to the biology analysis of recombinating.

It is biological to use the following step to analyze, as the total carotene that produces in the inventive method used yeast: by ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

Typical specimen preprocessing reason the following step constitutes: use polar organic solvent such as acetone or alcohol (as methyl alcohol) or ether to extract total lipid; Saponification; In alternate distribution; From more separating nonpolar epiphase and chromatogram the low phasic property derivative of polar.

For analysis, solvent is sent robot system [Gilson, Inc.USA, 3000W.Beltline Highway, Middleton, the WI] realization that can comprise single syringe valve Gilson 232XL and 402 2S1V diluters with the taking-up of aliquots containig by use.For saponification reaction, with potassium hydroxide water-ethanolic soln (4 water: 1 ethanol) join in each pipe, add the 3ml octanol then of 3ml50%.Saponification reaction is handled and can followingly be carried out: at room temperature pipe is placed on IKA HS 501 horizontal oscillator tubes [Labworld-online, Inc., Wilmington, NC], shook static then about 1 hour 15 hours with the speed that per minute 250 changes.

After saponification reaction, supernatant is diluted with 0.24ml methyl alcohol.For guaranteeing uniformity of sample, under pressure, add methyl alcohol.Use the 0.25ml syringe, can take out the aliquots containig of 0.1ml and be transferred in the HPLC pipe and analyze.

Analyze for HPLC, used Hewlett Packard 1100HPLC, it has been equipped with quaternary pump pump, vacuum outgas system, six logical introduction valves, thermoregulator self-actuated sampler, column oven and electric diode array detector [Agilent Technologies, can be from Ultra Scientific Inc., 250Smith Street, North Kingstown, RI obtains].Post can be the Waters YMC30 that has with the Material card sleeve column, 5-micron, 4.6 * 250mm[Waters, 34Maple Street, Milford, MA].The solvent of moving phase can be with 81 stable methyl alcohol of 0.2%BHT (2,6-di-t-butyl-4-cresols): 4 water: 15 tetrahydrofuran (THF)s (THF).Inject 20 μ l.Flow velocity with 1.7ml/ minute under 30 ℃ carries out the equipotential separation.Absorption measurement peak by the 447nm place.

For selected wild-type and the mutant sample of from the screening step, identifying, measure carotenoid compositions.The petal sample can be stored in-80 ℃ of refrigerators until identifying mutant.Can sample is moving dried, and the exsiccant tissue can be stored in-80 ℃ of guidance preparation analyses in the presence of argon.

Extraction step can carry out under ruddiness.Dry petal is ground and sieve with the No.40 mesh sieve.Accurately weighing grinds the weight of sample and is transferred in the red volumetric flask of 100ml.In sample, add 500 μ l H ₂O was with mixture vortex vibration 1 minute.Adding 30ml extracts solvent (10ml hexane+7ml acetone+6ml raw spirit+7ml toluene) and bottle was vibrated 10 minutes down at 160 rev/mins.

For saponification reaction, in bottle, add 2ml 40%KOH methanol solution, the vortex vibration is 1 minute then.Bottle is placed 56 ℃ of water-baths 20 minutes.Connect condenser to prevent solvent loss.Use the condenser that is connected in the dark with sample cooling 1 hour.After the cooling, add the 30ml hexane, and bottle was vibrated 10 minutes with 160 rev/mins.

Diluted sample after will vibrating with 10% metabisulfite solution is to 100ml, and thermal agitation 1 minute.Sample was placed the dark place at least 30 minutes.Take out the aliquots containig of 35ml mutually from the upper strata of about 50ml and be transferred to the sample cup.In bottle, add the 30ml hexane again, then with 160 rev/mins of vibrations 10 minutes.After about 1 hour, the upper strata is mixed mutually.Analyze for HPLC, the 10ml aliquots containig is dry and be stored in-80 ℃ in the presence of argon under nitrogen.

HPLC equipment is Alliance 2690, it is equipped with cooling self-actuated sampler, post well heater Waters Photodiode Array 996 detectors (Waters Corp., 34Maple StreetMilford, MA 01757). separation can (3m, 2.0 * 150mm) realize with having YMC30 post with the Material card sleeve column.Standard can be from ICC Indorespective fine chemicalsSomerville, New Jersey 088876 and DHI-Water ﹠amp; Environment, DK-2970Horsholm, Denmark obtains.

Exsiccant mutant sample can be resuspended in tetrahydrofuran (THF) and the methyl alcohol to cumulative volume 200l and filter, and contrast can additionally not concentrate.Carotenoid can separate with gradient method.Initial gradient condition can be 90% methyl alcohol: 5% water: 5% methyl tertiary butyl ether, flow velocity 0.4ml/min.From 0 to 15 minute, moving phase can become 80 methyl alcohol from initial condition: 5 water: 15 methyl tertiary butyl ethers from 15 to 60 minutes, are changed to 20 methyl alcohol: 5 water: 75 methyl tertiary butyl ethers.For later 10 minutes, the moving phase condition can be back to initial condition, and with column equilibration 10 minutes.Column temperature can be maintained at 27 ℃ and flow velocity and maintain 0.4ml/min.Injection 10l.Most peaks can 450nm measure and can from 286,348,400 and 472nm extract passage and add extra area.

[0459.0.24.24] if desired and expectation can use suitable resin to carry out more chromatographic step subsequently.Advantageously, can use so-called RPHPLC to be further purified carotene.Can use acetonitrile/water or chloroform/acetonitrile mixture as elutriant.If desired, can use same resin or other chromatography resin to repeat these chromatographic steps.The technician is familiar with the selection of suitable chromatography resin and they the effective use for specific molecular to be purified.

[0460.0.0.0] seen in disclosing of [0460.0.0.24] these paragraphs

[0461.0.24.24] embodiment 10: clone SEQ ID NO:34228 is used for expressing plant

[0462.0.0.0] seen in disclosing of [0462.0.0.24] these paragraphs

[0463.0.24.24] passes through pcr amplification SEQ ID NO:34228 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.24] to [0466.0.0.24]: see that [0464.0.0.0] is to [0466.0.0.0]

[0466.1.0.24] under the situation of using the Herculase enzyme to increase, the pcr amplification circulation is as follows: 94 ℃, and 2-3 minute, 1 circulation; 94 ℃, 30 seconds, 55-60 ℃, 30 seconds, 72 ℃, 5-10 minute, 25-30 circulation; 72 ℃, 10 minutes, 1 circulation; 4 ℃.

[0467.0.24.24] selects following primer sequence for genes of SEQ ID NO:34228:

I) forward primer (SEQ ID NO:34320)

Ii) reverse primer (SEQ ID NO:34321)

Perhaps as Table III the 7th row, 278-289 is capable or the primer of 637-641 shown in capable be used for selected at Table III the 5th row, 278-289 is capable or the capable described gene of 637-641.

[0468.0.0.24] to [0479.0.0.24]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.0.24.24] embodiment 11: express the generation of the transgenic plant of SEQ ID NO:34228

[0481.0.0.24] to [0513.0.0.24]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.24.24] alternatively, as Deli, J. and Molnar, P.Paprika carotenoids:Analysis, isolation, structure eucidation.Curr.Org.Chem.6,1197-1219 (2004) or Fraser, P.D., Pinto, M.E., Holloway, D.E. and Bramley, P.M.Technical advance:application of high-performance liquidchromatography with photodiode array detection to the metabolicprofiling of plant isoprenoids.Plant J.24,551-558 detects carotene described in (2000).

The different plants of being analyzed the results are shown in following table 1:

Table 1

??ORF	Metabolite	Method	??Min	??Max
					??b0730	Isopentenylpyrophosphate	??LC	??1.41	??2.15
??b1829	β-Hu Luobusu	??LC	??1.33	??1.74
					??b1926	Isopentenylpyrophosphate	??LC	??1.37	??1.55
??b2211	Isopentenylpyrophosphate	??LC	??1.46	??1.48
					??b2699	β-Hu Luobusu	??LC	??1.43	??1.76
??b3172	Isopentenylpyrophosphate	??LC	??1.35	??1.74
					??b4129	Isopentenylpyrophosphate	??LC	??1.34	??1.63
??YBR089C-A	β-Hu Luobusu	??LC	??1.52	??2.08
					??YDR316W	β-Hu Luobusu	??LC	??1.28	??1.52
??YDR407C	Isopentenylpyrophosphate	??LC	??1.54	??3.14
					??YDR513W	β-Hu Luobusu	??LC	??1.39	??1.43
??YLL013C	β-Hu Luobusu	??LC	??1.43	??1.50
					??b0970	Isopentenylpyrophosphate	??LC	??1.36	??2.17
??b0481	Isopentenylpyrophosphate	??LC	??1.35	??1.39
					??b1736	Isopentenylpyrophosphate	??LC	??1.51	??2.37
??b1738	Isopentenylpyrophosphate	??LC	??1.63	??2.60
					??b3160	Isopentenylpyrophosphate	??LC	??1.35	??1.47

[0515.0.24.24] to [0552.0.0.24]: see that [0515.0.0.0] is to [0552.0.0.0]

[0552.1.0.24]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

??ORF	Metabolite	??Min	??Max
				??b1829	β-Hu Luobusu	??1.40	??1.76

Table 2 shows that β-Hu Luobusu has increased in the genetic modification maize plant of expressing Escherichia coli nucleic acid sequence b1829.

In maize plant Escherichia coli protein b1829 have the heat shock protein of protease activity or situation that the activity of its homologue is enhanced under, preferably, give the fine chemicals β-Hu Luobusu and be increased between 40% and 76%.

[00552.2.0.24] sees [00552.2.0.0]

[0553.0.24.24]

1. produce the method for carotene and/or IPP, it comprises:

(a) improving in non-human being or its one or more parts or producing activity as protein shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or its function equivalent; With

(b) in allowing described biology, cultivate this biology under the condition of generation carotene and/or IPP.

2. produce the method for carotene and/or IPP, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is as polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, and described nucleic acid molecule is given in biology or its part carotene and/or IPP quantity increases;

B) contain just like Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of carotene and/or IPP quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of carotene and/or IPP quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that carotene in biology or its part and/or IPP quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives in biology or its part carotene and/or IPP quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part carotene and/or IPP quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in consensus sequence polypeptide and give biology or its part in carotene and/or IPP quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give carotene and/or the increase of IPP quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or bonded carotene and/or IPP.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or the bonded carotene and/or the IPP of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is as polypeptide or its fragment shown in Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable, and described nucleic acid molecule gives that carotene quantity increases in biology or its part;

B) contain just like Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 the nucleic acid molecule of the nucleic acid molecule shown in capable;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of carotene and/or IPP quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of carotene and/or IPP quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that carotene in biology or its part and/or IPP quantity increase with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises the nucleic acid molecule that as primer shown in Table III the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable or primer amplifier nucleic acid molecule from cDNA library or genomic library is obtained by using, and gives in biology or its part carotene and/or IPP quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give in biology or its part carotene and/or IPP quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 capable shown in consensus sequence polypeptide and give biology or its part in carotene and/or IPP quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give carotene and/or the increase of IPP quantity in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from as sequence shown in Table I A or IB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 row and/or the 278th, 280,281,283,284 and/or 287 row and/or 637-641 are capable by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding as claim 6 described in, thus this polypeptide by one or more amino acid be different from as Table II A or IIB the 5th or 7 row, the 279th, 282,285,286,288 and/or 289 are gone and/or the 278th, 280,281,283,284 and/or 287 go and/or 637-641 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in carotene and/or IPP quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving polypeptide that in biology or its part carotene and/or IPP quantity increase coded by the nucleic acid molecule of claim 6 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps linolic acid level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by the carotene that will measure and/or IPP level or expression of polypeptides level and standard linolic acid or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify and to give the method that carotene in plant or the microorganism and/or IPP produce the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of carotene and/or IPP quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of expression of polypeptides of carotene in biology or its part and/or the increase of IPP quantity and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify and to give the method that carotene in the cell and/or IPP produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that carotene and/or IPP increase after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce carotene and/or IPP;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) carotene and/or the IPP level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give carotene and/or the raising of IPP level in the host cell after it is expressed with wild-type.

20. identify and to give the method that carotene in the cell and/or IPP produce the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that carotene in biology or its part and/or IPP quantity or level improves after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce carotene and/or IPP;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) carotene and/or the IPP level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give carotene and/or the raising of IPP level in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify the nucleic acid molecule of giving carotene and/or IPP increase after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of regulation and control carotene and/or IPP level at biology.

25. makeup, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of any one described method of claim 1-5, claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20, wherein carotene is β-Hu Luobusu.

27. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make, and plant is anti-oxidant coerces.

28. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make, and plant is anti-causes oxidative stress or suppress carotenoid synthetic weedicide.

29. the purposes of the results material of the agonist of identifying according to claim 17, the plant of claim 16 or plant tissue, claim 16 or the host cell of claim 10 to 12, it is used to produce make-up composition.

[0554.0.0.24] summary: see [0554.0.0.0]

Produce the method for fine chemicals

[0000.0.0.25] the present invention relates to other method and this paper embodiment described below that as described below being used to produces fine chemicals in another embodiment.

[0001.0.0.0] seen in disclosing of [0001.0.0.25] these paragraphs

[0002.0.25.25] carbohydrate is to have polyhydric aldehydes or ketones compound.Many carbohydrates formula (CH2O) n that sees service; Some also comprise nitrogen, phosphorus or sulphur.Carbohydrate is divided into monose, oligosaccharides and polysaccharide.Monose or simple sugars are made up of a single poly-hydroxy aldehydes or ketones unit.Monose more than 4 carbon tends to have ring structure.Oligosaccharides is by the short chain monosaccharide units that links together by glycosidic link or residue, 2-19 unit forms usually.Polysaccharide is the sugared polymer that comprises more than about 20 monosaccharide units, and some polysaccharide have hundreds and thousands of units.Some polysaccharide are linear chains; And other is branched.Carbohydrate be known as sugar (saccharide) if or they be known as sugar (sugar) in relative hour.In the present invention, carbohydrate refers to all above-mentioned carbohydrates, for example monose, preferred fructose, glucose, inositol, semi-lactosi, pectinose, wood sugar or other pentoses or hexose; Oligosaccharides, preferred disaccharides such as sucrose, lactose or trisaccharide such as raffinose; Perhaps polysaccharide such as starch or Mierocrystalline cellulose.

[0003.0.25.25] carbohydrate is a abundantest class organic compound of finding in the living organism.They are main sources of plant and animal metabolisable energy.Except sugar and starch satisfies important trophism, the carbohydrate function also relates to energy storage (for example starch or glycogen), signal transmission (by glycoprotein and glycolipid, blood group determinant for example), give neural system, muscle and in fact be that whole cell fueling, carbohydrate are the parts (in gene, mRNA, tRNA, rrna) of nucleic acid, and become the recognition site on the cell surface and participate in the signal transmission as the cell surface marker thing, and glycolipid and glycoprotein are also as structured material cell wall constituent (Mierocrystalline cellulose) for example.

[0004.0.25.25] glucose is also referred to as dextrose, be the sugar the most widely that distributes in plant and animal circle, and the sugar that is present in the blood is called " blood sugar ".It occupies middle position in the metabolism of plant, animal and many microorganisms.Glucose is rich in the potential energy, and therefore is good fuel; The breakdown of glucose metabolism produces ATP in body.Glucose is stored as high molecular polymer for example starch or glycogen, perhaps is converted into lipid acid.It still is general precursor, and the metabolic intermediate of huge amount can be provided for biological building-up reactions.Based on its many-sided feature, glucose is used for nutrition and medicine.Fructose is also referred to as sinistrose or " Fruit candy ", is most important ketose.Fructose is hexose and is reducing sugar.Fructose is used as sweetener by the diabetics, the glucose level because fructose does not raise, even the glucose level that also can not raise when a large amount of the use.Fructose and glucose are the main carbohydrate components of honey.Those hexoses still are the main component of many oligosaccharides and polysaccharide (as sucrose, raffinose, stachyose, trehalose, starch, Mierocrystalline cellulose or dextran) in addition.Modal disaccharides is that (sucrose, b-D-fructose furyl glycosyl-a-D-glucopyranoside, sucrose, beet sugar, sugar is meant the sucrose that commerce can get to sucrose on narrow meaning, this means that sucrose is the sugar that is commonly referred to " sugar "), it comprises 6 carbon sugar D-glucose and D-fructose.Sucrose is formed by plant, rather than is formed by animal.Sucrose is photosynthetic main intermediate product; In some plants, sucrose is sugar is transported to other position of plant materials from leaf principal mode.In Mammals, sucrose is the essential composition of blood, and its content in blood remains on maintenance level.Sucrose also is very essential for the normal function of brain cell and central nervous system.Sugar is widely regarded as the source of glycogen, and glycogen is the material that is delivered to liver, heart and muscle.Sucrose is one of the most widely used foodstuff products, and is disaccharides main in most diet.It is present in honey, maple sugar, fruit, strawberry and vegetables.It can be used as liquid or crystalline saccharose or adds in the foodstuff products as Nulomoline.Prepare from sugar-cane or sugar beet at commercial sucrose.Sucrose can provide a large amount of desired function characteristics by feeding produce product, comprises fragrance, mouthfeel and the ability that changes between noncrystalline and crystalline state.High concentration sucrose is a natural antiseptic agent, and it determines gel formation process, gives the viscosity of product necessity.Sucrose is the coarse raw materials of caramel, pigment or the like.Sucrose still is fabulous fermentation raw material, and this is for fermentation industry (comprising a large amount of non-food product industry-pharmaceutical industries) particularly advantageous.Exist 8 oh groups that the theoretical possibility of very a large amount of sucrose derivatives is provided at sucrose molecules.Sucrose derivative is used for the production of stain remover, emulsifying agent (sucrose+lipid acid) and tackiness agent (eight acetic acid sucrose) by manufacturing enterprise.

Sucrose is carbohydrate in the plant, as is found in the particularly precursor of the raffinose family of the oligosaccharides of beans of many plant seeds.This family comprises trisaccharide raffinose, tetrose stachyose and pentose verbascose.The oligosaccharides of raffinose series is main component (Shallenberger etc., J.Agric.Food Chem., 9,1372 of many edible beans; 1961).Raffinose (beta-D-fructofuranose base-6-O-α-D-galactopyranose base-α-D-glucopyranoside, melitriose, gossypose, melitose), it is made up of the sucrose that contains semi-lactosi, wherein semi-lactosi is connected to by its C-4 atom on 1 of residue of fructose, and be considered to be in the non-structure carbohydrate in the vegitabilia abundance be only second to second of sucrose and enrich carbohydrate.It can be ubiquitous, is ubiquitous at least in higher plant.Raffinose accumulates edible part in many important economic farm crop with remarkable quantity.Example comprises that soybean, sugar beet, cotton, rape and all main edible pulse family farm crop comprise soybean, pea, French beans and lupine.

Important crucial intermediate was inositol (hexanaphthene-1,2,3,4,5, a 6-hexanol) during raffinose and stachyose formed, prevailing cyclitol.Inositol is many different sorts plant-growths and grows required base substance.In addition, it can also be as the biosynthetic precursor of phytic acid, inositol also is used for uronide and pentose biosynthesizing, and it also is present in the phosphoinositide of plant cell membrane, and other complicated plant lipid comprises in the Phosphoric acid glycerol esters brain acid amides (glycophosphoceramides).And inositol still is other natural precursor that has the inositol isomer, and in the species AD HOC of whole plants circle, many these inositols distribute and form methyl ether.Important somatomedin in the inositol.Most carbohydrates that occurring in nature is found exist with the polysaccharide form, thereby polysaccharide is the polymkeric substance generation high molecular of intermediate.Polysaccharide is also referred to as saccharan, aspect the identity of its monosaccharide unit that repeats to exist, the length aspect of its chain, connect the type aspect of each unitary key, and differing from each other aspect branch degree.

Starch is the polysaccharide of most worthy.Normal native starch is made up of the mixture of 15-30% amylose starch and 70-85% amylopectin.Be the linear polymer of anhydroglucose unit on the amylose starch structure, between 40 000 and 340 000, its chain comprises the 250-2000 anhydroglucose unit to molecular weight greatly.Amylopectin is considered to be made up of the anhydroglucose chain with many tapping points; Molecular weight can be up to 80 000 000.

Starch is most important, abundant, the most digestible food polysaccharide.It is present in leaf, stem, root, seed, fruit and the pollen of many higher plants as storing polysaccharide.Starch exists with particle isolating, partial crystallization, and its size, shape and gelatinization temperature depend on the plant origin of starch.The common food starch source is in seed (wheat, corn, paddy rice, barley) and root (potato, cassava/cassava).Starch has been modified the functional performance with the improvement expection, and is added into food as foodstuff additive with relatively small amount.

Another kind of important polysaccharide is a Mierocrystalline cellulose.Mierocrystalline cellulose is modal polysaccharide, and scientist estimates the annual synthetic 1,000,000 tons of Mierocrystalline celluloses that surpass of plant.Mierocrystalline cellulose forms the cell walls of plant.It remain monose-glucose-the 3rd polymer.Mierocrystalline cellulose is different from starch and glycogen, because glucose unit forms two-dirnentional structure, combines by hydrogen bond and polymer nearby, thereby gives more stability of molecule.Single " cellulosic fibre " may comprise 10000 anhydroglucose unit of as many as.In Mierocrystalline cellulose, each fiber molecule bunchy is arranged, and therefore forms so-called dimension protofibril, and it finally forms the net spline structure of " the dense braiding " of cellulosic molecule.Strong coherency between each cellulosic fibre is owing to there is the strong hydrogen bonding of enormous quantity.

Mierocrystalline cellulose is the main polysaccharide of grass, leaf and trees, and is described to be included in about 50% of all biological carbon found on our celestial body.It is the basic material of crude substance such as timber, flax or cotton, and comprises long not branched fiber molecule.As vegetable fibre, Mierocrystalline cellulose can not be by human digestive, so Mierocrystalline cellulose can pass digestive tube and not be absorbed into body.Some animals such as milk cow and termite contain the bacterium of helpful their digest cellulose in its digestive tube.Even so, it is important that Mierocrystalline cellulose remains in human nutrition, because fiber is the necessary part of diet, a large amount of foods is provided and promotes intestinal peristalsis.

[0005.0.25.25] polysaccharide starch and Mierocrystalline cellulose are most important coarse raw materials in glucose industry and the commercial production.In common process, starch or Mierocrystalline cellulose are become glucose by acid or enzymic hydrolysis.Fructose also produces from starch usually, forms dextrose syrup by amylase is transformed, and handles with isomerase subsequently, and causing conversion of glucose is fructose.

Sucrose commercial be to obtain from the squeezing juice of sugar-cane or sugar beet.

There is (being found in for example sugarcane, beet sirup and almond shell) in inositol or as six phosphoric acid that are called phytic acid (being found in for example corn steep liquor) at occurring in nature or with its free form.Comprise from corn steep liquor industrial purification phytic acid and to use calcium deposit, use the strong acid hydrolysis subsequently.Comprise with acid treatment with by pillar (US 5,482,631) or utilize ion-exchange (US4,482,761) to separate inositol from plant milk extract separated free form inositol.

Mierocrystalline cellulose is very important Industrial products.As above disclosed, Mierocrystalline cellulose is as the coarse raw materials that produces monose.In addition Mierocrystalline cellulose also can be used for making paper, textiles, plastics, explosive substance, wrapping material (

), feed, food and leavened prod.Mierocrystalline cellulose mainly obtains by acid or basic hydrolysis.

Starch exists with big bunch or particle in cell.These pearl starchs are by microscopic granulometric composition, and it is different with shape to depend on its size of plant origin.Particle is water insoluble in room temperature.Known have very a large amount of methods to be used to extract starch.For example heating comprises the grinding starch slurry of vegetable material, and grain expansion also finally breaks thus, and starch molecule is dispensed into solution.In liquefaction step, long-chain starch further is degraded to less branch or linear unit (dextrin) by α-Dian Fenmei.Big metering method has been described and has been used for starch is converted into starch hydrolysate, and for example maltose, glucose or specified syrup agent is perhaps as sweetener or as the precursor of other carbohydrate such as fructose.The enzymatic hydrolysis process that is used for pearl starch is hydrolyzed into the Zulkovsky starch hydrolysate is disclosed in US 20050042737.

[0006.0.25.25] carbohydrate has main effect in human and animal's diet, the energy that accounts for about 40-75% is taken in.Its most important nutritive property is a digestibility.In them some become monose by the enzymic hydrolysis in people's gastro-intestinal system, and monose is absorbed into small intestine and enters the sugar metabolism approach.Can being digested of other by some animal.

Carbohydrate, fat and protein are the production capacity nutrition of animal-feed.In the general diet of domestic animal, the level of carbohydrate is 70-80%.For example the food of pig is mainly based on cereal, and cereal comprises the nutraceutical major portion of the essential energy supply of pig.

From the global food needs that increases gradually, because the arable land operability of growing world population and atrophy simultaneously, it is important improving food and the feeding quality operability that particularly some must nutrition (preferred carbohydrate, preferred polysaccharide such as starch or Mierocrystalline cellulose and/or monose such as fructose, glucose and/or inositol and/or trisaccharide such as raffinose and/or disaccharides such as sucrose).Nutrition improvement can help to satisfy the demand to improving the quality in food and the feed.Modern agriculture biotechnology (relate to application cell and molecular engineering will encode the DNA of anticipated shape be passed to food and feed farm crop) is effectively replenishing of traditional method, can satisfy global food and feed demand.

[0007.0.25.25] in addition, physico-chemical property such as viscosity and combination water and ionic ability are different between different cereal grasss.As a result, different cereal characteristics influence digestion and the fermentation in the gi tract by different way, and microorganism species.Gastrointestinal disturbance constitutes the subject matter of humans and animals health.

Need suitable diet formation and food or the preferred cereal of forage component, beans or fruit, they promote favourable intestinal environment and prevent gastro intestinal disorders.Therefore the quality that improves food and animal-feed is a task important in food and the fodder industry.This is inevitable, because the carbohydrate that for example is present in plant and some microorganisms is limited for the Mammals supply.Particularly advantageous for the quality of food and animal-feed is carbohydrate spectrum in the balanced diet as far as possible because on the specific concentrations some excessive carbohydrates only have some or seldom or do not have an active effect.

The genetically modified plant that [0008.0.25.25] has the trophic spectrum of improvement is well known in the art.US 20030070192 discloses the DNA expression cassette that changes the sugar alcohol that transforms plant.US5,908,975 relate to the synthetic and polymeric method of accumulation fructose in transgenic plant by using-system specificity promoter and leaf bubble target sequence selective expression bacterium fructosyltransferase gene.WO89/12386 has described and has been used for producing glucose and the polymeric method of fructose the transgenic Fructus Lycopersici esculenti plant.Described the stress tolerance sequence and comprise protein such as Galactinol synthase (GOLS) and raffinose synthase (RAFS) in US 20050055748, they raise in stress reaction and cause producing raffinose.US 6,887, and 708 nucleotide sequences that provide coding to have the polypeptide of Arabidopis thaliana GIGANTEA gene function, this gene allow operation starch accumulation process in plant.In US20050091716, disclose the embryo of (for example Aa/Bb) the genotype heterozygosis that has two or more wild type genes and had the endosperm of this genoid genotype heterozygosis, and caused plant to have the cereal of the normal starch route of synthesis of change.

[0009.0.25.25] even so, new enzymic activity or direct or indirect instrumentality still constantly need be provided, and the alternative approach that therefore need have advantageous feature is used for producing carbohydrate biological as genetically modified organism, preferred polysaccharide such as starch or Mierocrystalline cellulose and/or monose such as fructose, glucose and/or inositol and/or trisaccharide such as raffinose and/or disaccharides such as sucrose or its precursor.

[0010.0.25.25] another problem is the seasonal variation of plant carbohydrate components, and best harvesting time and periodic problem exist simultaneously.

[0011.0.25.25] is in order to continue to guarantee the high quality of food and animal-feed, be necessary that with balance mode with one or more carbohydrates, preferred polysaccharide such as starch or Mierocrystalline cellulose and/or monose such as fructose, glucose and/or inositol and/or trisaccharide such as raffinose and/or disaccharides such as sucrose are added into suitable biology.

[0012.0.25.25] therefore, still be starved of one or more codings and participate in carbohydrate, the biosynthetic enzyme of preferred polysaccharide such as starch or Mierocrystalline cellulose and/or monose such as fructose, glucose and/or inositol and/or trisaccharide such as raffinose and/or disaccharides such as sucrose or other proteinic suitable gene, and make and might not form unwanted byproduct with specific these products of generation of technical scale.In the screening that is used for biosynthetic gene or instrumentality, two characteristics are particularly important especially.On the one hand, but the highest intrinsic energy carbohydrate of the acquisition of needs improvement forever, the method for preferred polysaccharide such as starch or Mierocrystalline cellulose and/or monose such as fructose, glucose and/or inositol and/or trisaccharide such as raffinose and/or disaccharides such as sucrose; On the other hand, reduce the byproduct that produces in the production process as far as possible.The carbohydrate that is added further advantageously influences micropopulation by the growth and/or the activity of selective stimulating beneficial bacteria.Another aspect is the remarkable reduction of production and manufacturing cost, is not only particularly sweetener industry of nutrition, also comprises agricultural and makeup and health industries.

[0013.0.0.0] seen in disclosing of [0013.0.0.25] these paragraphs

[0014.0.25.25] therefore, in the first embodiment, the present invention relates to produce the method for fine chemicals, fine chemicals is carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose thus.Therefore, in the present invention, term " fine chemicals " refers to " carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose " as used herein.In addition, term " fine chemicals " refers to comprise carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably composition of the fine chemicals of sucrose as used herein.

[0015.0.25.25] in one embodiment, term " carbohydrate " or " fine chemicals " or " each fine chemicals " meaning is meant that at least a active chemical compound of carbohydrate that has, described carbohydrate activity are preferably selected from polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose.In preferred embodiments, term " fine chemicals " or term " carbohydrate " or term " each fine chemicals " meaning is meant that at least a active chemical compound of carbohydrate that has, described carbohydrate activity are selected from " carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose ".

Usually, the contents of saccharide of the increase meaning is meant the total glucides content of increase.Yet the contents of saccharide of increase can also specifically refer to have the change of the active above-claimed cpd content of carbohydrate, must increase and need not total glucides content.In preferred embodiments, term " fine chemicals " meaning is meant free form or its salt or ester-formin or is bonded to the carbohydrate, preferred polysaccharide of acylglycerol form, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose.

[0015.1.22.22] can measure anhydroglucose content to the mensuration of polysaccharide of the present invention, preferred starch and content of cellulose.If as preparation sample as described in the embodiment and measure, this compound is the analyte that indication polysaccharide of the present invention, preferred starch and Mierocrystalline cellulose exist.

[0016.0.25.25] therefore, the method that the present invention relates to may further comprise the steps:

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0252, b1430, b1693, b3231, YER174C, b0050, b1539, b3919 and/or b4232 protein or have by Table II the 5th or 7 row, 290-294 and/or 604-607 capable shown in the protein of the coded peptide sequence of nucleic acid molecule; With

(b) in allowing described biology, produce fine chemicals be starch and/or Mierocrystalline cellulose or comprise starch and/or the condition of cellulosic fine chemicals under make biological growth.

Perhaps

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0161, b0695, b1708, b1926, b2597, b2664, b4239, b4327, b0124, b0149, b1318, b1463, b2491, b3260, b3578, b3619 and/or b4122 protein or have by Table II the 5th or 7 row, 295-302 and/or 608-616 capable shown in the protein of the coded peptide sequence of nucleic acid molecule; With

(b) in allowing described biology, produce under the condition that fine chemicals is fructose or the fine chemicals that comprises fructose and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0161, b1708, b1926, b2599, b4239, YJL072C, b1463, b1736, b2491, b3578 and/or b3619 protein or have by Table II the 5th or 7 row, 303-308 and/or 617-621 capable shown in the protein of the coded peptide sequence of nucleic acid molecule; With

(b) producing fine chemicals in allowing described biology is to make biological growth under glucose or the condition that comprises the glucose fine chemicals.

Perhaps

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0138, b0290, b2023, b2699, b3172, b3430, b4129, YBR204C, YDR112W, YGR261C, YIL150C, YJL099W, YOR044W, YOR350C, b1463, b1961, b4074 and/or YHR072W-A protein or have by Table II the 5th or 7 row, the protein of the coded peptide sequence of nucleic acid molecule shown in 309-322 and/or 622-625 are capable; With

(b) in allowing described biology, produce under the condition that fine chemicals is inositol or the fine chemicals that comprises inositol and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0161, b0730, b1701, b1886, b2664, b2699, b3601, YBR184W, YGR261C and/or b3578 protein or have protein by the coded peptide sequence of nucleic acid molecule shown in Table II the 5th or 7 row, 323-331 and/or 626 row; With

(b) in allowing described biology, produce under the condition that fine chemicals is raffinose or the fine chemicals that comprises raffinose and make biological growth.

Perhaps

(a) in non-human being or its one or more parts, improve or produce one or more following activity of proteins: b0019 and/or b4239 protein or have by Table II the 5th or 7 row, 332-333 capable shown in the protein of the coded peptide sequence of nucleic acid molecule; With

(b) in allowing described biology, produce under the condition that fine chemicals is sucrose or the fine chemicals that comprises sucrose and make biological growth.

Therefore, the present invention relates to the method that may further comprise the steps:

(a) in non-human being or one or more part, improve or produce one or more activity of proteins, described protein has Table II the 3rd row, 290-333 is capable and/or 604-626 capable shown in activity of proteins, perhaps have by Table I the 5th or 7 the row, 290-333 is capable and/or 604-626 capable shown in nucleic acid molecule encoded polypeptide sequence and

(b) in allowing described biology, produce fine chemicals be carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably make biological growth under the condition of sucrose.

[0016.1.25.25] therefore, term " fine chemicals " looks like in one embodiment and is meant " polysaccharide, preferred starch and/or Mierocrystalline cellulose " or its homologue relevant with Table I-IV 290-294 and/or the capable listed full sequence of 604-607; And the meaning is meant " monose, preferred fructose " or its homologue relevant with Table I-IV 295-302 and/or the capable listed full sequence of 608-616 in one embodiment; And the meaning is meant " monose, preferred glucose " or its homologue relevant with Table I-IV 303-308 and/or the capable listed full sequence of 617-621 in one embodiment; And the meaning is meant " monose, preferably myo-inositol " or its homologue relevant with Table I-IV 309-322 and/or the capable listed full sequence of 622-625 in one embodiment; And the meaning is meant " trisaccharide, preferred raffinose " or its homologue relevant with Table I-IV 323-331 and/or the listed full sequence of 626 row in one embodiment; And the meaning is meant " disaccharides, preferably sucrose " or its homologue relevant with the capable listed full sequence of Table I-IV 332-333 in one embodiment.

Therefore, in one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 295th, 303 and 323 row and/or relevant " fructose ", " glucose " and " raffinose " of the 614th and the 620 and 626 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 611st, 617 and 622 row and/or relevant " fructose ", " glucose " and " inositol " of the 611st and the 617 and 622 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 297th and 304 row and/or the 612nd and 619 row and/or the 614th and 620 row and/or the 615th and 621 row and/or relevant " fructose " and " glucose " of the 611st and 617 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 298th and relevant " fructose " and " glucose " of the 305 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 300th and relevant " fructose " and " raffinose " of the 327 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 301st, 307 and relevant " fructose ", " glucose " and " sucrose " of the 333 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 312nd and relevant " inositol " and " raffinose " of the 328 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 318th and relevant " inositol " and " raffinose " of the 331 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 611st and relevant " inositol " and " fructose " of the 622 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 617th and relevant " inositol " and " glucose " of the 622 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 614th and relevant " fructose " and " raffinose " of the 626 listed full sequences of row; In one embodiment, term " fine chemicals " meaning is meant and Table I-IV the 620th and relevant " glucose " and " raffinose " of the 626 listed full sequences of row.

Therefore, term " fine chemicals " can refer to " starch and/or Mierocrystalline cellulose ", " fructose ", " glucose ", " inositol ", " raffinose " and/or " sucrose " according to environment and context.For the meaning of illustrating term " fine chemicals " is meant " starch and/or Mierocrystalline cellulose ", " fructose ", " glucose ", " inositol ", " raffinose " and/or " sucrose ", also can use term " each fine chemicals ".

[0017.0.25.25] to [0018.0.25.25]: see that [0017.0.0.0] is to [0018.0.0.0]

The method that [0019.0.25.25] produces each fine chemicals advantageously causes the generation of each fine chemicals to strengthen.Term " enhancing " or " raising " meaning are to compare each fine chemicals generation to improve 10% at least with following defined reference, 20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, be more preferably 150%, 200%, 300%, 400% or 500%, wherein said " comparing " meaning with following defined reference make finger for example with such biophase ratio, promptly should biology do not exist having as Table II the 3rd row, protein active shown in the capable and/or 604-626 of 290-333 is capable or by as Table I the 5th or 7 row, the activity of proteins of the coded protein active of nucleic acid molecule was carried out above-mentioned modification shown in the capable and/or 604-626 of 290-333 was capable.

[0020.0.25.25] is surprisingly found out that, and be at least a as going for starch and/or cellulosic Table II the 3rd row, the 294th; The 308th row for glucose; 316-322 and/or 625 row for inositol; Each fine chemicals content that yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana shown in capable is given the plant that transforms for the 330-331 of raffinose increases; And/or it is at least a as capable for starch and/or cellulosic Table II the 3rd row, 290-293 and/or 604-607; 295-302 and/or 608-616 for fructose are capable; 303-307 and/or 617-621 for glucose are capable; 309-315 and/or 622-624 for inositol are capable; 323-329 and/or 626 row for raffinose; Each fine chemicals content that e. coli k12 protein transgene expression in Arabidopis thaliana shown in capable is given the plant that transforms for the 332-333 of sucrose increases.

Therefore, be surprisingly found out that fructose, glucose or raffinose (or each fine chemicals) content that e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 295th, 303 and 323 row and/or the 614th and 620 and 626 row is given the plant that transforms increases.Therefore, in one embodiment, described protein or its homologue are used to produce fructose; In one embodiment, described protein or its homologue are used to produce glucose; In one embodiment, described protein or its homologue are used to produce raffinose; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from fructose, glucose and raffinose.

Therefore, be surprisingly found out that fructose, glucose or inositol (or each fine chemicals) content that e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 5th row, the 611st, 617 and 622 row is given the plant that transforms increases.Therefore, in one embodiment, described protein or its homologue are used to produce fructose; In one embodiment, described protein or its homologue are used to produce glucose; In one embodiment, described protein or its homologue are used to produce inositol; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from fructose, glucose and inositol.

Therefore, be surprisingly found out that, Table II the 5th row, the 297th and 304 row and/or the 298th and 305 row and/or and/or the 612nd and 619 row and/or the 614th and 620 row shown in e. coli k12 protein transgene expression in Arabidopis thaliana give fructose or the increase of glucose (or each fine chemicals) content of the plant that transforms.Therefore, in one embodiment, described protein or its homologue are used to produce fructose; In one embodiment, described protein or its homologue are used to produce glucose; In one embodiment, described protein or its homologue are used to produce the fine chemicals that one or more are selected from fructose and glucose.

Therefore, be surprisingly found out that fructose or raffinose (or each fine chemicals) content that e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 300th and 327 row is given the plant that transforms increase.Therefore, in one embodiment, described protein or its homologue are used to produce fructose; In one embodiment, described protein or its homologue are used to produce raffinose; In one embodiment, described protein or its homologue are used to produce fructose and raffinose.

Therefore, be surprisingly found out that fructose, glucose or sucrose (or each fine chemicals) content that e. coli k12 protein transgene expression in Arabidopis thaliana shown in Table II the 301st, 307 and 333 row is given the plant that transforms increases.Therefore, in one embodiment, described protein or its homologue are used to produce fructose; In one embodiment, described protein or its homologue are used to produce glucose; In one embodiment, described protein or its homologue are used to produce sucrose; In one embodiment, described protein or its homologue are used to produce fructose and glucose; In one embodiment, described protein or its homologue are used to produce fructose and sucrose; In one embodiment, described protein or its homologue are used to produce dextrose plus saccharose; In one embodiment, described protein or its homologue are used to produce fructose, dextrose plus saccharose.

Therefore, be surprisingly found out that inositol or raffinose (or each fine chemicals) content that yeast saccharomyces cerevisiae protein transgene expression in Arabidopis thaliana shown in e. coli k12 protein shown in Table II the 312nd and 328 row or Table II the 3rd row, the 318th and 331 row is given the plant that transforms increase.Therefore, in one embodiment, described protein or its homologue are used to produce inositol; In one embodiment, described protein or its homologue are used to produce raffinose; In one embodiment, described protein or its homologue are used to produce inositol and raffinose.

[0021.0.0.0] seen in disclosing of [0021.0.25.25] these paragraphs

The sequence of [0022.0.25.25] e. coli k12 b0019 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Na+/H+ antiport albumen.Therefore, in one embodiment, the inventive method comprises as shown herely having an active gene product of Na+/H+-exchanger matter nhaA superfamily from e. coli k12, preferably have the protein of Na+/H+ antiport protein-active or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be carbohydrate, the sucrose of preferably free or combining form.

The sequence of e. coli k12 b0138 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity be defined as be the supposition umbrella portion sample (fimbrial-like) adhesin protein.Therefore, in one embodiment, the inventive method comprises the purposes of the gene product with e. coli k12 umbrella portion sample adhesin protein active as shown here or its homologue (gene product or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of e. coli k12 b0161 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be pericentral siphon serine protease (heat shock protein).Therefore, in one embodiment, the inventive method comprises the active gene product of Helicobacterium serine protease superfamily that has as shown here, preferably have the active protein of pericentral siphon serine protease (heat shock protein) or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. fructose, glucose and/or the raffinose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b0252 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity is still undefined, but its for have the conservative protein matter of the unknown function of similarity with helicase and ligase enzyme.Therefore, in one embodiment, the inventive method comprises as shown here have helicase or the active gene product of ligase enzyme, the purposes that preferably has the active protein of e. coli k12 b0252 or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. the starch and/or the Mierocrystalline cellulose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b0290 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be receptor protein.Therefore, in one embodiment, the inventive method comprises the active gene product of intestinal bacteria yagW protein superfamily that has as shown here, the purposes that preferably has the active protein of receptor protein or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of e. coli k12 b0695 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be sensibility histidine kinase in the two-pack regulator control system.Therefore, in one embodiment, the inventive method comprises the active gene product of sensibility histidine kinase superfamily that has as shown here, preferably have the active protein of sensibility histidine kinase in the two-pack regulator control system or the purposes of its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred fructose of free or combining form.

The sequence of e. coli k12 b0730 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be that succinic thiokinase operon transcriptional and acyl are replied regulatory gene.Therefore, in one embodiment, the inventive method comprises the active gene product of transcriptional GntR superfamily that has as shown here, preferably has the purposes that succinic thiokinase operon transcriptional and acyl are replied the active protein of regulatory gene or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred raffinose of free or combining form.

The sequence of e. coli k12 b1430 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be S-adenosine-L-methionine-dependent methyltransgerase of giving the tellurous acid resistance.Therefore, in one embodiment, the inventive method comprises the active gene product of hemagglutinin hag1 superfamily that has as shown here, the purposes that preferably has protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) of S-adenosine-L-methionine-dependent methyl transferase activity of giving the tellurous acid resistance, it is used for producing fine chemicals in biological or its part as described, i.e. the starch and/or the Mierocrystalline cellulose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b1693 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the 3-dehydroquinate dehydratase.Therefore, in one embodiment, the inventive method comprises the active gene product of 3-dehydroquinate dehydratase superfamily that has as shown here, the purposes that preferably has the active protein of 3-dehydroquinate dehydratase or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. the starch and/or the Mierocrystalline cellulose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b1701 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the coenzyme A dependency ligase enzyme with Photinus pyralis LUC-sample ATP enzymatic structure territory.Therefore, in one embodiment, the inventive method comprises as shown here have 4-Coumarate-CoA ligase or the active gene product of Acetate-CoA ligase superfamily, the purposes that preferably has active or its homologue (protein or its homologue from e. coli k12 for example as shown here) of protein coenzyme A dependency ligase enzyme, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred raffinose of free or combining form.

The sequence of e. coli k12 b1708 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be lipoprotein.Therefore, in one embodiment, the inventive method comprises the active gene product of protein HI1314 superfamily that has as shown here, the purposes that preferably has the active protein of lipoprotein or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, preferably free or combining form fructose and/or glucose.

The sequence of e. coli k12 b1886 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be methyl acceptor chemotactic protein matter II or aspartic acid transmitter acceptor.Therefore, in one embodiment, the inventive method comprises the active gene product of methyl acceptor chemotactic protein matter superfamily that has as shown here, the purposes that preferably has protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) of methyl acceptor chemotactic protein matter II or aspartic acid transmitter receptor active, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred raffinose of free or combining form.

The sequence of e. coli k12 b1926 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be flagellin matter fliT.Therefore, in one embodiment, the inventive method comprises the purposes with the active gene product of flagellin matter fliT or its homologue (protein or its homologue from e. coli k12 for example as shown here) as shown here, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, preferably free or combining form fructose and/or glucose.

The sequence of e. coli k12 b2023 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the glutamine acid amides transferring enzyme subunit of imidzoleglycerol phosphate synthase heterodimer.Therefore, in one embodiment, the inventive method comprises that tool as shown here has the active gene product of amide transferase hisH superfamily of trpG homology, the purposes that preferably has the active protein of glutamine acid amides transferring enzyme subunit or its homologue (protein or its homologue from e. coli k12 for example as shown here) of imidzoleglycerol phosphate synthase heterodimer, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of e. coli k12 b2597 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the rrna correlation factor.Therefore, in one embodiment, the inventive method comprises the active gene product of pseudomonas putida (Pseudomonas putida) protein rpoX superfamily that has as shown here, the purposes that preferably has the active protein of rrna correlation factor or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred fructose of free or combining form.

The sequence of e. coli k12 b2599 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be bifunctional enzyme: chorismate mutase (N-end) and prephenate dehydratase (C-end).Therefore, in one embodiment, the inventive method comprises the active gene product of as shown here having " the pheA bifunctional enzyme superfamily with prephenate dehydratase homology ", preferably has bifunctional enzyme: the purposes of chorismate mutase (N-end) and prephenate dehydratase (C-end) active protein or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred glucose of free or combining form.

The sequence of e. coli k12 b2664 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be to have the transcription repressor (GntR family) of DNA in conjunction with wing spirane structure territory.Therefore, in one embodiment, the inventive method comprises the active gene product of transcriptional gabP superfamily that has as shown here, what preferably have supposition has the purposes of DNA in conjunction with transcription repressor (GntR family) active protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) in wing spirane structure territory, it is used for producing fine chemicals in biological or its part as described, i.e. the fructose and/or the raffinose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b2699 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be DNA chain exchange and the recombinant protein with proteolytic enzyme and nuclease.Therefore, in one embodiment, the inventive method comprises the active gene product of recombinant protein recA superfamily that has as shown here, the purposes that preferably has " exchange of DNA chain and recombinant protein " active protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) with proteolytic enzyme and nuclease, it is used for producing fine chemicals in biological or its part as described, i.e. the inositol and/or the raffinose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b3172 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be arginyl succsinic acid synthetic enzyme.Therefore, in one embodiment, the inventive method comprises the active gene product of arginyl succsinic acid synthetic enzyme superfamily that has as shown here, the purposes that preferably has protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) of arginyl succsinic acid synthase activity, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of e. coli k12 b3231 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be 50S ribosomal subunit protein matter L13.Therefore, in one embodiment, the inventive method comprises the active gene product of intestinal bacteria ribosomal protein L13 superfamily that has as shown here, the purposes that preferably has the active protein of 50S ribosomal subunit protein matter L13 or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. the starch and/or the Mierocrystalline cellulose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b3430 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the Cori ester adenylyl transferase.Therefore, in one embodiment, the inventive method comprises the active gene product of Cori ester adenylyl transferase superfamily that has as shown here, the purposes that preferably has the active protein of Cori ester adenylyl transferase or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of e. coli k12 b3601 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the transcription repressor that utilizes for N.F,USP MANNITOL.Therefore, in one embodiment, the inventive method comprises the purposes of the active protein of transcription repressor as shown here or its homologue (protein or its homologue from e. coli k12 for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred raffinose of free or combining form.

The sequence of e. coli k12 b4129 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be (derivable) Methionin tRNA synthetic enzyme.Therefore, in one embodiment, the inventive method comprises the active gene product of Lysine-tRNA ligase superfamily that has as shown here, the purposes that preferably has protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) of Methionin tRNA synthase activity, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of e. coli k12 b4239 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be trehalose-6-P lytic enzyme.Therefore, in one embodiment, the inventive method comprises as shown here from having " the alpha-glucosidase superfamily with α-Dian Fenmei core homology " active gene product, the purposes that preferably has protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) of trehalose-6-P hydrolytic enzyme activities, it is used for producing fine chemicals in biological or its part as described, i.e. fructose, glucose and/or the sucrose of carbohydrate, preferably free or combining form.

The sequence of e. coli k12 b4327 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the HTH-type transcriptional (LysR family) with periplasmic binding protein matter structural domain.Therefore, in one embodiment, the inventive method comprises the active gene product of as shown here having " the protein b2409 superfamily with α-Dian Fenmei core homology ", the purposes that preferably has " HTH-type transcriptional (LysR family) " active protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) with periplasmic binding protein matter structural domain, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred fructose of free or combining form.

The sequence of e. coli k12 b4327 has been published in Blattner etc., Science277 (5331), 1453-1474,1997, and its activity to be defined as be the HTH-type transcriptional (LysR family) with periplasmic binding protein matter structural domain.Therefore, in one embodiment, the inventive method comprises the active gene product of as shown here having " the protein b2409 superfamily with α-Dian Fenmei core homology ", the purposes that preferably has " HTH-type transcriptional (LysR family) " active protein or its homologue (protein or its homologue from e. coli k12 for example as shown here) with periplasmic binding protein matter structural domain, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred fructose of free or combining form.

The sequence of yeast saccharomyces cerevisiae YBR184w has been published in Goffeau, and Science 274 (5287), 546-547, and 1996, and in Feldmann, EMBO J., 13,5795-5809,1994, and its activity does not characterize yet.Therefore, in one embodiment, the inventive method comprises the active gene product of protein YBR184w superfamily that has as shown here, the purposes that preferably has the active protein of protein YBR184w or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred raffinose of free or combining form.

The sequence of yeast saccharomyces cerevisiae YBR204C has been published in Goffeau, and Science 274 (5287), 546-547,1996 and Feldmann, EMBO J., 13,5795-5809,1994, and its activity to be defined as be peroxysome lipase.Therefore, in one embodiment, the inventive method comprises the purposes of the protein with peroxysome lipase activity as shown here or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

Yeast saccharomyces cerevisiae YDR112w (accession number S69752), its activity is still undefined.Therefore, in one embodiment, the inventive method comprises the purposes with the active protein of YDR112w or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here) as shown here, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of yeast saccharomyces cerevisiae YER174C has been published in Dietrich, Nature 387 (6632 supplementary issue), 78-81,1997, and Goffeau, Science 274 (5287), 546-547,1996, and its activity to be defined as be the reactive gsh dependency of hydrogen peroxide and superoxide radical oxydo-reductase.Therefore, in one embodiment, the inventive method comprises the active gene product of Trx superfamily that has as shown here, preferably have the protein of the reactive gsh dependency of hydrogen peroxide and superoxide radical oxidoreductase activity or the purposes of its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. the starch and/or the Mierocrystalline cellulose of carbohydrate, preferably free or combining form.

The sequence of yeast saccharomyces cerevisiae YGR261C has been published in Tettelin, Nature 387 (6632 supplementary issue), 81-84,1997, and Goffeau, Science 274 (5287), 546-547,1996, and its activity to be defined as be clathrin assembling complex body β adaptin composition.Therefore, in one embodiment, the inventive method comprises the β-active gene product of adaptin superfamily that has as shown here, the purposes that preferably has the clathrin assembling complex body β active protein of adaptin composition or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, be carbohydrate, the inositol and/or the raffinose of preferably free or combining form.

The sequence of yeast saccharomyces cerevisiae YIL150C has been published in Goffeau st al., Science 274 (5287), 546-547,1996 and Churcher etc., Nature 387 (6632 supplementary issue), 84-87,1997, and its activity to be defined as be that the S phase (DNA synthetic) is initial or finish necessary chromobindins.Therefore, in one embodiment, the inventive method comprises that as shown here having the S phase (DNA synthetic) is initial or finishes the purposes of the active protein of necessary chromobindins or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of yeast saccharomyces cerevisiae YJL072C has been published in Goffeau, A., and Science 274 (5287), 546-547,1996 and Galibert, F., EMBO be (9) J.15,2031-2049,1996, and its activity to be defined as be the subunit that chromosomal DNA duplicates required GINS complex body.Therefore, in one embodiment, the inventive method comprises the active gene product of yeast saccharomyces cerevisiae membranin YJL072c superfamily that has as shown here, preferably has the purposes that chromosomal DNA duplicates the active protein of subunit or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here) of required GINS complex body, it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred glucose of free or combining form.

The sequence of yeast saccharomyces cerevisiae YJL099W has been published in Goffeau, A., and Science 274 (5287), 546-547,1996 and Galibert, F., EMBO be (9) J.15,2031-2049,1996, and its activity to be defined as be the protein that participates in chitin biosynthesizing and/or its adjusting.Therefore, in one embodiment, the inventive method comprises the gene product with the protein active that participates in chitin biosynthesizing and/or its adjusting as shown here or the purposes of its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of yeast saccharomyces cerevisiae YOR044W has been published in Dujon, Nature 387 (6632 supplementary issue), and 98-102,1997, and Goffeau, Science 274 (5287), 546-547, and 1996, and its activity does not characterize yet.Therefore, in one embodiment, the inventive method comprises the active gene product of yeast saccharomyces cerevisiae membranin YOR044w superfamily that has as shown here, the purposes that preferably has the active protein of YOR044W or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of yeast saccharomyces cerevisiae YOR350C has been published in Dujon, Nature 387 (6632 supplementary issue), and 98-102,1997, and Goffeau, Science 274 (5287), 546-547, and 1996, and its activity does not characterize yet.Therefore, in one embodiment, the inventive method comprises the active gene product of yeast saccharomyces cerevisiae MNE1 protein superfamily that has as shown here, the purposes that preferably has the active protein of YOR350C or its homologue (protein or its homologue from yeast saccharomyces cerevisiae for example as shown here), it is used for producing fine chemicals in biological or its part as described, i.e. carbohydrate, the preferred inositol of free or combining form.

The sequence of b0050 (accession number NP_414592) e. coli k12 has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the conservative protein matter of potential participation protein-protein interaction.Therefore, in one embodiment, the inventive method comprises from the active gene product of the colibacillary apaG of having protein superfamily, preferably have the active protein of conservative protein matter of potential participation protein-protein interaction or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be starch and/or Mierocrystalline cellulose, particularly increase the starch and/or the cellulosic quantity of starch and/or Mierocrystalline cellulose, preferably free or combining form.

The sequence of e. coli k12 b0124 (accession number NP_414666) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be Hexose phosphate dehydrogenase.Therefore, in one embodiment, the inventive method comprises from colibacillary having an active gene product of Hexose phosphate dehydrogenase (Pyrroloquinoline quinone) superfamily, the purposes that preferably has protein or its homologue of glucose dehydrogenase activity, it is used for producing fine chemicals in biological or its part as described, be fructose, particularly increase fructose, the preferred quantity of the fructose of free or combining form.

The sequence of e. coli k12 b0149 (accession number NP_414691) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be bi-functional penicillin-conjugated protein 1b: glycosyltransferase (N-end); Transpeptidase (C-end).Therefore, in one embodiment, what the inventive method comprised has penicillin-active gene product of conjugated protein 1B superfamily from colibacillary, preferably has " bi-functional penicillin-conjugated protein 1b: glycosyltransferase (N-end); Transpeptidase (C-end) " purposes of active protein or its homologue, it is used for producing fine chemicals in biological or its part as described, i.e. and fructose particularly increases fructose, the preferred quantity of the fructose of free or combining form.

The sequence of e. coli k12 b1318 (accession number NP_415834) has been published in Blattner, and Science 277 (5331), 1453-1474, and 1997, and its activity is defined as HUCEP-8 (ATP-binding cassette superfamily).Therefore, in one embodiment, the inventive method comprises the active gene product of inner membrane protein malK (having the ATP-binding cassette homology) superfamily that has as shown here, preferably from colibacillary purposes with the active protein of HUCEP-8 (ATP-binding cassette superfamily) or its homologue, it is used for producing fine chemicals in biological or its part as described, promptly fructose, particularly increase the quantity of the fructose of free or combining form.

The sequence of e. coli k12 b1463 (accession number NP_415980) has been published in Blattner, and Science 277 (5331), 1453-1474, and 1997, and its activity is defined as N-hydroxy aromatic amine O-Transacetylase.Therefore, in one embodiment, the inventive method comprises the active gene product of arylamine acetyl transferase superfamily that has as shown here, preferably have the protein of N-hydroxy aromatic amine O-acetyltransferase activity or a purposes of its homologue from colibacillary, it is used for producing fine chemicals in biological or its part as described, be fructose and/or glucose and/or inositol, particularly increase fructose, particularly increase glucose, particularly increase inositol, particularly increase fructose and glucose, particularly increase fructose and inositol, particularly increase glucose and inositol, particularly increase fructose and glucose and inositol, preferably free or the fructose of combining form and/or the quantity of glucose and/or inositol.

The sequence of e. coli k12 b1539 (accession number NP_416057) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be NADP dependency L-Serine/L-not-threonate dehydrogenase.Therefore, in one embodiment, the inventive method comprise have ribitol dehydrogenase, the active gene product of short-chain alcohol dehydrogenase homology superfamily, preferably from colibacillary have NADP dependency L-Serine/L-not-purposes of the active protein of threonate dehydrogenase or its homologue, it is used for producing fine chemicals in biological or its part as described, be starch and/or Mierocrystalline cellulose, particularly increase the starch and/or the cellulosic quantity of starch and/or Mierocrystalline cellulose, preferably free or combining form.

The sequence of e. coli k12 b1736 (accession number NP_416250) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be cellobiose/arbutin/saligenin sugar-specific protein PEP dependency phosphotransferase.Therefore, in one embodiment, the inventive method comprises having " phosphotransferase system lactose specific enzymes II; factor III superfamily " active gene product, preferably have the cellobiose/arbutin/saligenin sugar-protein of specific protein PEP dependency phosphate transferase activity or a purposes of its homologue from colibacillary, it is used for producing fine chemicals in biological or its part as described, be glucose, particularly increase glucose, the preferred quantity of the glucose of free or combining form.

The sequence of e. coli k12 b1961 (accession number NP_416470) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be DNA cytosine methylation enzyme.Therefore, in one embodiment, the inventive method comprises the active gene product of (cytosine specific) EcoRII superfamily that has site-specific methyltransferase, preferably have the protein of DNA cytosine methylation enzymic activity or a purposes of its homologue from colibacillary, it is used for producing fine chemicals in biological or its part as described, be inositol, particularly increase inositol, the preferred quantity of the inositol of free or combining form.

The sequence of e. coli k12 b2491 (accession number NP_416986) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be transcriptional activation agent (EBP family) with σ 54 interactional hydrogenase 4 genetic expressions.Therefore, in one embodiment, the inventive method comprises having " RNA polymerase Sigma Factors interaction domain homology; transcriptional activation agent fhlA; the nif-specificity is regulated protein; reply regulatory gene homology superfamily " active gene product, preferably have and transcriptional activation agent (EBP family) the active protein of σ 54 interactional hydrogenase 4 genetic expressions or the purposes of its homologue from colibacillary, it is used for producing fine chemicals in biological or its part as described, be fructose and/or glucose, particularly increase fructose, particularly increase glucose, particularly increase fructose and glucose, preferably free or the fructose of combining form and/or the quantity of glucose.

The sequence of e. coli k12 b3260 (accession number NP_417726) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be tRNA-dihydrouridine synthase B.Therefore, in one embodiment, the inventive method comprises having the active gene product of transcriptional yacF superfamily, preferably from colibacillary purposes with the active protein of tRNA-dihydrouridine synthase B or its homologue, it is used for producing fine chemicals in biological or its part as described, be fructose, particularly increase fructose, the preferred quantity of the fructose of free or combining form.

The sequence of e. coli k12 b3578 (accession number YP_026232) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity be defined as be the supposition the movement system composition.Therefore, in one embodiment, the inventive method comprises as shown here from the movement system composition of colibacillary supposition or the purposes of its homologue, it is used for producing fine chemicals in biological or its part as described, be fructose and/or glucose and/or raffinose, particularly increase fructose, particularly increase glucose, particularly increase raffinose, particularly increase fructose and glucose, particularly increase fructose and raffinose, particularly increase glucose and raffinose, particularly increase fructose, glucose and raffinose, preferably free or the fructose of combining form and/or the quantity of glucose and/or raffinose.

The sequence of e. coli k12 b3619 (accession number NP_418076) has been published in Blattner etc., Science 277 (5331), 1453-1474,1997, and its activity to be defined as be NAD (P) associativity ADP-L-glycerine-D-mannoheptose-6-epimerase.Therefore, in one embodiment, the inventive method comprises having " ADP glycerine mannoheptose 6-epimerase; UDP glucose 4-epimerase homology superfamily " active gene product, preferably from colibacillary purposes with NAD (P) associativity ADP-L-glycerine-D-mannoheptose-active protein of 6-epimerase or its homologue, it is used for producing fine chemicals in biological or its part as described, be fructose and/or glucose, particularly increase fructose, particularly increase glucose, particularly increase fructose and glucose, preferred free or the fructose of combining form and/or the quantity of glucose.

The sequence of e. coli k12 b3919 (accession number NP_418354) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be triosephosphate isomerase.Therefore, in one embodiment, the inventive method comprises has the active gene product of triosephosphate isomerase superfamily, preferably from colibacillary purposes with the active protein of triosephosphate isomerase or its homologue, it is used for producing fine chemicals in biological or its part as described, be starch and/or Mierocrystalline cellulose, particularly increase starch and/or Mierocrystalline cellulose, the starch and/or the cellulosic quantity of preferably free or combining form.

The sequence of e. coli k12 b4074 (accession number NP_418498) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be the biological albumen that takes place of cytochrome c-type.Therefore, in one embodiment, the inventive method comprises having the active gene product of nrfE protein superfamily, preferably have the biological protein of protein-active or a purposes of its homologue of taking place of cytochrome c-type from colibacillary, it is used for producing fine chemicals in biological or its part as described, be inositol, particularly increase inositol, the preferred quantity of the inositol of free or combining form.

The sequence of e. coli k12 b4122 (accession number NP_418546) has been published in Blattner etc., and Science 277 (5331), 1453-1474,1997, and its activity to be defined as be FURAMIC ACID B (fumaric acid I class hydratase).Therefore, in one embodiment, the inventive method comprises having iron dependency fumarate hydratase and/or the active gene product of iron dependency tartrate dehydratase α chain homology superfamily, preferably from colibacillary purposes with FURAMIC ACID B (fumaric acid I class hydratase) active protein or its homologue, it is used for producing fine chemicals in biological or its part as described, be fructose, particularly increase fructose, the preferred quantity of the fructose of free or combining form.

E. coli k12 b4232 (accession number: NP_416986) be published in Blattner, Science 277 (5331), 1453-1474, and 1997, and its activity is defined as fructose-1.Therefore, in one embodiment, the inventive method comprises the purposes from colibacillary " fructose-1; 6-diphosphatase " or its homologue as shown here, it is used for producing fine chemicals in biological or its part as described, be methionine(Met), particularly increase the quantity of the methionine(Met) of free or combining form.In one embodiment, in the methods of the invention, superfamily " fructose-diphosphatase " protein, preferably has C-compound and sugar metabolism, C-compound and carbohydrate utilization, energy, glycolysis-and glyconeogenesis, plastid, the active protein of light compositing, more preferably have " fructose-1; 6-diphosphatase " active activity of proteins and be enhanced or produce, for example the activity from colibacillary protein or its homologue is enhanced or produces.Therefore, in one embodiment, in the methods of the invention, the activity of " fructose-1; 6-diphosphatase " or its homologue is enhanced to give in biology or its part and produces fine chemicals, be starch and/or Mierocrystalline cellulose, particularly increase the starch and/or the cellulosic quantity of free or combining form.

The sequence of yeast saccharomyces cerevisiae YHR072W-A (accession number NP_058135) has been published in Goffeau etc., Science 274 (5287), 546-547,1996, and its activity to be defined as be the small nucleolar ribonucleoprotein particulate moiety that comprises H/ACA-type snoRNA (it is that preceding 18SrRNA carries out pseudouracilization and processing is required).Therefore, in one embodiment, the inventive method comprises the purposes of the small nucleolar ribonucleoprotein particulate moiety that comprises H/ACA-type snoRNA from yeast saccharomyces cerevisiae as shown here (it is that preceding 18S rRNA carries out pseudouracilization and processing is required) or its homologue, it is used for producing fine chemicals in biological or its part as described, be inositol, particularly increase inositol, the preferred quantity of the inositol of free or combining form.

The homologous compound (=homologue) of [0023.0.25.25] gene product of the present invention can be from any biology, as long as homologous compound is given activity as herein described, particularly given each fine chemicals quantity or content and increase.

In one embodiment, Table II the 3rd is listed as, goes for starch and/or the cellulosic the 294th respectively; For the 308th row of glucose, for the 316-322 of inositol and/or 625 row, for the homologue of one of polypeptide shown in the 330-331 of raffinose and/or 626 row is to have same or similar active homologue, particularly, the active raising given each fine chemicals content increase in the biology.In one embodiment, homologue be have Table I or II the 7th row, respectively for starch and/or cellulosic the 294th row, for the 308th row of glucose, for the 316-322 of inositol and/or 625 row, for the homologue of sequence shown in the 330-331 of raffinose and/or 626 row.

In one embodiment, Table II the 3rd row, respectively for starch and/or Mierocrystalline cellulose the 294th row, for the 308th row of glucose, for the 316-322 of inositol and/or 625 row, for the homologue of one of polypeptide shown in the 330-331 of raffinose and/or 626 row from eukaryote.In one embodiment, homologue is from fungi.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 294th row is from Ascomycota.In one embodiment, the homologue of polypeptide shown in Table II the 3rd row, the 294th row is from yeast.In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic the 294th row, for the 308th row of glucose, for the 316-322 of inositol and/or 625 row, for the homologue of polypeptide shown in the 330-331 of raffinose and/or 626 row from the yeast guiding principle.In one embodiment, Table II the 3rd row, to be respectively for starch and/or cellulosic the 294th row, for the 308th row of glucose, for the 316-322 of inositol and/or 625 row, for the homologue of polypeptide shown in the 330-331 of raffinose and/or 626 row be homologue from Saccharomycetes.In one embodiment, Table II the 3rd row, to be respectively for starch and/or Mierocrystalline cellulose the 294th row, for the 308th row of glucose, for the 316-322 of inositol and/or 625 row, for the homologue of polypeptide shown in the 330-331 of raffinose and/or 626 row be to have same or similar activity and from the homologue of Saccharomycetaceae.In one embodiment, Table II the 3rd row, to be respectively for starch and/or cellulosic the 294th row, for the 308th row of glucose, for the 316-322 of inositol and/or 625 row, for the homologue of polypeptide shown in the 330-331 of raffinose and/or 626 row be to have same or similar activity and from the homologue of yeast guiding principle.

In one embodiment, Table II the 3rd row, 290-293 capable shown in the homologue of one of polypeptide be to have same or similar active homologue.Particularly, the active raising given the preferred carbohydrate of each fine chemicals in the biology, preferred starch and/or content of cellulose increase.In one embodiment, homologue be have Table I or II the 7th row, 290-293 capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, 290-293 capable shown in the homologue of one of polypeptide from bacterium.In one embodiment, Table II the 3rd row, 290-293 capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, 290-293 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, 290-293 capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, 290-293 capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, 290-293 capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of one of polypeptide be to have same or similar active homologue.Particularly, the active raising given the preferred carbohydrate of each fine chemicals in the biology, preferred fructose content increase.In one embodiment, homologue be have Table I or II the 7th row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of sequence.In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of one of polypeptide from bacterium.In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of polypeptide from Proteobacteria.In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of γ-distortion Gammaproteobacteria.In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of polypeptide from the enterobacteria order.In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of polypeptide be homologue from enterobacteriaceae.In one embodiment, Table II the 3rd row, be respectively for starch and/or cellulosic 290-293 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-307 of glucose and/or 617-621 is capable, for the 309-315 of inositol and/or 622-624 is capable, capable for the 323-329 of raffinose, for the 332-333 of sucrose capable shown in the homologue of polypeptide be to have same or similar activity and from the homologue of Escherichia.

[0023.1.25.25] Table II the 3rd row, 290-333 is capable and/or 604-626 capable shown in the homologue of polypeptide can be by Table I the 7th row, 290-333 is capable and/or 604-626 capable shown in nucleic acid molecule encoding polypeptide or Table II the 7th row, 290-333 is capable and/or 604-626 capable shown in polypeptide.

[0024.0.0.0] seen in disclosing of [0024.0.0.9] these paragraphs

[0025.0.25.25] is according to the present invention, if from the beginning (the de novo) of protein or polypeptide expression active or that increase can directly or indirectly cause biology or its part, carbohydrate in the preferred described biomass cells, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level increases, then described protein or polypeptide have " activity of proteins of the present invention ", promptly have for example Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in activity of proteins.In preferred embodiments, protein or polypeptide have Table II the 3rd row, respectively for starch and/or cellulosic 290-294 and/or 604-607 is capable, for the 295-302 of fructose and/or 608-616 is capable, for the 303-308 of glucose and/or 617-621 is capable, for the 309-322 of inositol and/or 622-625 is capable, for the 323-331 of raffinose and/or 626 row, for the 332-333 of sucrose capable shown in proteinic above-mentioned extra activity.In this manual, if this kind protein or polypeptide still have Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in any one proteinic biology or the enzyme activity, if promptly with Table II the 3rd row, for starch and/or cellulosic the 294th row, the 308th row for glucose, 316-322 and/or 625 row for inositol, for yeast saccharomyces cerevisiae protein shown in the 330-331 of raffinose and/or 626 row or Table II the 3rd row, capable for starch and/or cellulosic 290-293 and/or 604-607,295-302 and/or 608-616 for fructose are capable, 303-307 for glucose is capable, 309-315 and/or 622-624 for inositol are capable, 323-329 for raffinose is capable, for the 332-333 of sucrose capable shown in the e. coli k12 protein any one compare, it has 10% of initial enzymic activity at least, preferred 20%, preferred especially 30%, most preferably 40%, then active the or preferred biologic activity of the nucleic acid molecule of this kind protein or polypeptide or encode this kind protein or polypeptide or sequence is identical or similar.

[0025.1.0.25]：see[0025.1.0.0]

[0026.0.0.25] to [0033.0.0.25]: see that [0026.0.0.0] is to [0033.0.0.0]

[0034.0.25.25] is preferably, and be only different with research object of the present invention aspect the cytoactive of polypeptide of the present invention with reference to, contrast or wild-type, and these differences are because nucleic acid molecule level for example of the present invention improves, or due to the raising of the specific activity of polypeptide of the present invention.For example, it has Table II the 3rd row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, protein shown in 332-333 is capable, or by Table I the 5th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the protein of nucleic acid molecule encoding shown in 332-333 is capable, or its homologue is (as Table I the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, homologue shown in 332-333 is capable) active protein expression level or active aspect difference, and its difference aspect biological chemistry or genetics reason.Therefore it shows each fine chemicals quantity that increases.

[0035.0.0.25] to [0044.0.0.25]: see that [0035.0.0.0] is to [0044.0.0.0]

[0045.0.25.25] in one embodiment, at e. coli k12 protein b0019 or its homologue, for example under the situation that Na+/H+-exchanger matter nhaA superfamily activity of proteins is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 332nd row) of Na+/H+ antiport protein-active, give each fine chemicals, preferably sucrose be increased in 22% and 55% or more between.

In one embodiment, at e. coli k12 protein b0138 or its homologue, for example have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 309th row) of umbrella portion sample adhesin protein active, give each fine chemicals, preferably myo-inositol be increased in 32% and 46% or more between.

In one embodiment, at e. coli k12 protein b0161 or its homologue, for example under the situation that the activity of Helicobacterium serine protease superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of pericentral siphon serine protease (heat shock protein) (for example shown in Table I the 5th or 7 row, the 295th, 303 and 323 row), give each fine chemicals, preferred fructose (82%-1155%), glucose (86%-338%) and/or raffinose (128%-197%) be increased in 82% and 1155% or more between.

In one embodiment, at e. coli k12 protein b0252 or its homologue, for example have with helicase and ligase enzyme and have under the activity situation about being enhanced of b0252 protein active protein (for example shown in Table I the 5th or 7 row, the 290th row) of similarity, give each fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 37% and 53% or more between.

In one embodiment, at e. coli k12 protein b0290 or its homologue, for example under the situation that the activity of intestinal bacteria yagW protein superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of receptor protein (for example shown in Table I the 5th or 7 row, the 310th row), give each fine chemicals, preferably myo-inositol be increased in 23% and 50% or more between.

In one embodiment, at e. coli k12 protein b0695 or its homologue, for example have under the situation that the active activity of proteins of sensibility histidine kinase superfamily homology is enhanced, preferably, have under the situation that the activity of the active protein of sensibility histidine kinase in the reactive two-part regulator control system (for example shown in Table I the 5th or 7 row, the 296th row) is enhanced, give each fine chemicals, preferred fructose be increased in 69% and 1046% or more between.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example under the situation that the activity of transcriptional GntR superfamily is enhanced, preferably, have under the activity situation about being enhanced that succinic thiokinase operon transcriptional and acyl reply the active protein of regulatory gene (for example shown in Table I the 5th or 7 row, the 324th row), give each fine chemicals, preferred raffinose be increased in 93% and 616% or more between.

In one embodiment, at e. coli k12 protein b1430 or its homologue, for example under the situation that hemagglutinin hag1 superfamily activity of proteins is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 291st row) of the S-adenosine of the giving the tellurous acid resistance-L-methionine-dependent methyl transferase activity of supposition, give each fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 31% and 58% or more between.

In one embodiment, at e. coli k12 protein b1693 or its homologue, for example under the situation that the activity of 3-dehydroquinate dehydratase superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of 3-dehydroquinate dehydratase (for example shown in Table I the 5th or 7 row, the 292nd row), give each fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 34% and 116% or more between.

In one embodiment, at e. coli k12 protein b1701 or its homologue, for example have under the situation that 4-Coumarate-CoA ligase and/or the active protein active of Acetate-CoA ligase superfamily homology be enhanced, preferably, have under the activity situation about being enhanced of " coenzyme A dependency ligase enzyme " active protein (for example shown in Table I the 5th or 7 row, the 325th row) with Photinus pyralis LUC-sample ATP enzymatic structure territory, give each fine chemicals, preferred raffinose be increased in 57% and 958% or more between.

In one embodiment, at e. coli k12 protein b1708 or its homologue, for example under the situation that the activity of protein HI1314 superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of lipoprotein (for example shown in Table I the 5th or 7 row, the 297th and 304 row), give each fine chemicals, preferred fructose (77%-2664%) and/or glucose (62%-942%) be increased in 62% and 2664% or more between.

In one embodiment, at e. coli k12 protein b1886 or its homologue, for example under the situation that the activity of methyl acceptor chemotactic protein matter superfamily is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 326th row) of methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter receptor active, give each fine chemicals, preferred raffinose be increased in 64% and 391% or more between.

In one embodiment, at e. coli k12 protein b1926 or its homologue, for example have under the activity situation about being enhanced of the active protein of flagellin matter fliT (for example shown in Table I the 5th or 7 row, the 298th and 305 row), give each fine chemicals, preferred fructose (86%-193%) and/or glucose (63%-88%) be increased in 63% and 193% or more between.

In one embodiment, at e. coli k12 protein b2023 or its homologue, for example have under the situation that the activity of the amide transferase hisH superfamily of trpG homology is enhanced, preferably, have under the activity situation about being enhanced of the active protein of glutamine acid amides transferring enzyme subunit (for example shown in Table I the 5th or 7 row, the 311st row) of imidzoleglycerol phosphate synthase heterodimer, give each fine chemicals, preferably myo-inositol be increased in 31% and 110% or more between.

In one embodiment, at e. coli k12 protein b2597 or its homologue, for example under the situation that the activity of pseudomonas putida putative protein matter rpoX superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of rrna correlation factor (for example shown in Table I the 5th or 7 row, the 299th row), give each fine chemicals, preferred fructose be increased in 90% and 105% or more between.

In one embodiment, at e. coli k12 protein b2599 or its homologue, for example have under the situation that the activity of the pheA bifunctional enzyme superfamily of prephenate dehydratase homology is enhanced, preferably, have bifunctional enzyme: under the activity situation about being enhanced of chorismate mutase (N-end) and the active protein of prephenate dehydratase (C-end) (for example shown in Table I the 5th or 7 row, the 306th row), give each fine chemicals, preferred glucose be increased in 65% and 171% or more between.

In one embodiment, at e. coli k12 protein b2664 or its homologue, for example under the situation that the activity of transcriptional gabP superfamily is enhanced, preferably, have under the activity situation about being enhanced of " having the transcription repressor (GntR family) of DNA " active protein (for example shown in Table I the 5th or 7 row, the 300th and 327 row) in conjunction with wing spirane structure territory, give each fine chemicals, preferred fructose (77%-4086%) and/or raffinose (72%-1267%) be increased in 72% and 4086% or more between.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example under the situation that the activity of recombinant protein recA superfamily is enhanced, preferably, have under the situation that the activity of " DNA chain exchange and recombinant protein with proteolytic enzyme and nuclease " active protein (for example shown in Table I the 5th or 7 row, the 312nd and 328 row) is enhanced, give each fine chemicals, preferably myo-inositol (86%-690%) and/or raffinose (61%-2408%) be increased in 61% and 2408% or more between.

In one embodiment, at e. coli k12 protein b3172 or its homologue, for example under the situation that the activity of arginyl succsinic acid synthase superfamily is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 313rd row) of arginyl succsinic acid synthase activity, give each fine chemicals, preferably myo-inositol be increased in 26% and 144% or more between.

In one embodiment, at e. coli k12 protein b3231 or its homologue, for example under the situation that the activity of intestinal bacteria ribosomal protein L13 superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of 50S ribosomal subunit protein matter L13 (for example shown in Table I the 5th or 7 row, the 293rd row), give each fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 31% and 74% or more between.

In one embodiment, at e. coli k12 protein b3430 or its homologue, for example under the situation that the activity of Cori ester adenylyl transferase superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of Cori ester adenylyl transferase (for example shown in Table I the 5th or 7 row, the 314th row), give each fine chemicals, preferably myo-inositol be increased in 34% and 116% or more between.

In one embodiment, at e. coli k12 protein b3601 or its homologue, for example have under the situation that the activity of the active protein of transcription repressor that utilizes for N.F,USP MANNITOL (for example shown in Table I the 5th or 7 row, the 329th row) is enhanced, give each fine chemicals, preferred raffinose be increased in 84% and 135% or more between.

In one embodiment, at e. coli k12 protein b4129 or its homologue, for example under the situation that the activity of Lysine-tRNA ligase superfamily is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 315th row) of (derivable) Methionin tRNA synthase activity, give each fine chemicals, preferably myo-inositol be increased in 29% and 48% or more between.

In one embodiment, at e. coli k12 protein b4239 or its homologue, for example have under the situation that the activity of the alpha-glucosidase superfamily of α-Dian Fenmei core homology is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 301st, 307 and 333 row) of trehalose-6-P hydrolytic enzyme activities, give each fine chemicals, preferred fructose (321%-768%), glucose (100%-385%) and/or sucrose (64%-169%) be increased in 64% and 768% or more between.

In one embodiment, at e. coli k12 protein b4327 or its homologue, for example under the situation that the activity of protein b2409 superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of HTH-type transcriptional (LysR family) (for example shown in Table I the 5th or 7 row, the 302nd row) of periplasmic binding protein matter structural domain, give each fine chemicals, preferred fructose be increased in 97% and 275% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR184W or its homologue, for example under the situation that YBR184W protein superfamily-activity of proteins is enhanced, preferably, under the activity situation about being enhanced of YBR184W protein (for example shown in Table I the 5th or 7 row, the 330th row), give each fine chemicals, preferred raffinose be increased in 87% and 478% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR204C or its homologue, for example under the activity situation about being enhanced of peroxysome lipase protein (for example shown in Table I the 5th or 7 row, the 316th row), give each fine chemicals, preferably myo-inositol be increased in 27% and 192% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR112W or its homologue, for example under the activity situation about being enhanced of YDR112W protein (for example shown in Table I the 5th or 7 row, the 317th row), give each fine chemicals, preferably myo-inositol be increased in 41% and 90% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YER174C or its homologue, for example under the situation that the activity of Trx homology superfamily is enhanced, preferably, under the situation that the activity of the reactive gsh dependency of hydrogen peroxide and superoxide radical oxide-reductase protein matter (for example shown in Table I the 5th or 7 row, the 294th row) is enhanced, give each fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 34% and 84% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YGR261C or its homologue, for example under the situation that the activity of β-adaptin superfamily is enhanced, preferably, under the situation that the activity of clathrin assembling complex body β adaptin composition protein (for example shown in Table I the 5th or 7 row, the 318th and 331 row) is enhanced, give each fine chemicals, preferably myo-inositol (124%-698%) and/or raffinose (95%-2956%) be increased in 95% and 2956% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (for example shown in Table I the 5th or 7 row, the 319th row) is enhanced, give each fine chemicals, preferably myo-inositol be increased in 400% and 480% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL072C or its homologue, for example under the situation that the activity of yeast saccharomyces cerevisiae membranin YJL072c superfamily is enhanced, preferably, chromosomal DNA duplicates under the activity situation about being enhanced of protein subunit matter (for example shown in Table I the 5th or 7 row, the 308th row) of required GINS complex body, give each fine chemicals, preferred glucose be increased in 58% and 293% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL099W or its homologue, for example participate under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 320th row) of chitin biosynthesizing and/or its adjusting, give each fine chemicals, preferably myo-inositol be increased in 26% and 472% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR044W or its homologue, for example under the activity situation about being enhanced of yeast saccharomyces cerevisiae membranin YOR044w superfamily (for example shown in Table I the 5th or 7 row, the 321st row), give each fine chemicals, preferably myo-inositol be increased in 44% and 160% or more between.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR350C or its homologue, for example under the activity situation about being enhanced of yeast saccharomyces cerevisiae MNE1 protein superfamily (for example shown in Table I the 5th or 7 row, the 322nd row), give each fine chemicals, preferably myo-inositol be increased in 52% and 182% or more between.

At e. coli k12 protein b0050 or its homologue (shown in Table I the 5th or 7 row, the 604th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 38% and 89% or more between.

At e. coli k12 protein b0124 or its homologue (shown in Table I the 5th or 7 row, the 608th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred fructose be increased in 100% and 2926% or more between.

At e. coli k12 protein b0149 or its homologue (shown in Table I the 5th or 7 row, the 609th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred fructose be increased in 68% and 95% or more between.

At e. coli k12 protein b1318 or its homologue (shown in Table I the 5th or 7 row, the 610th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred fructose be increased in 103% and 199% or more between.

(be listed as at e. coli k12 protein b1463 or its homologue as Table I the 5th or 7, shown in the 611st and 617 and 622 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred fructose is increased between 92% and 453%, preferred glucose is increased between 57% and 618%, preferably myo-inositol is increased between 36% and 96%, preferred fructose and glucose are increased between 57% and 618%, preferred fructose and inositol are increased between 36% and 453%, preferred glucose and inositol are increased between 36% and 618%, preferred fructose and glucose and inositol be increased in 36% and 618% or more between.

At e. coli k12 protein b1539 or its homologue (shown in Table I the 5th or 7 row, the 605th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 39% and 92% or more between.

At e. coli k12 protein b1736 or its homologue (shown in Table I the 5th or 7 row, the 618th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred glucose be increased in 61% and 110% or more between.

At e. coli k12 protein b1961 or its homologue (shown in Table I the 5th or 7 row, the 623rd row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferably myo-inositol be increased in 25% and 58% or more between.

At e. coli k12 protein b2491 or its homologue (shown in Table I the 5th or 7 row, the 612nd and 619 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred fructose is increased between 123% and 278%, preferred glucose is increased in 81% and 289%, particularly increase fructose and glucose be increased in 81% and 289% or more between.

At e. coli k12 protein b3260 or its homologue (shown in Table I the 5th or 7 row, the 613rd row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred fructose be increased in 87% and 472% or more between.

(be listed as at e. coli k12 protein b3578 or its homologue as Table I the 5th or 7, shown in the 614th and 620 and 626 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred fructose is increased between 89% and 106%, preferred glucose is increased between 65% and 110%, preferred raffinose is increased between 55% and 84%, preferred fructose and glucose are increased between 65% and 110%, preferred glucose and raffinose are increased between 55% and 110%, preferred fructose and raffinose are increased in, between 55% and 106%, preferred fructose and glucose and raffinose be increased in 55% and 110% or more between.

At e. coli k12 protein b3619 or its homologue (shown in Table I the 5th or 7 row, the 615th and 621 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals in one embodiment, preferred fructose is increased between 61% and 111%, preferred glucose is increased between 60% and 119%, preferred fructose and glucose be increased in 60% and 119% or more between.

At e. coli k12 protein b3919 or its homologue (shown in Table I the 5th or 7 row, the 606th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 33% and 52% or more between.

At e. coli k12 protein b4074 or its homologue (shown in Table I the 5th or 7 row, the 624th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferably myo-inositol be increased in 25% and 50% or more between.

At e. coli k12 protein b4122 or its homologue (shown in Table I the 5th or 7 row, the 616th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred fructose be increased in 53% and 502% or more between.

At e. coli k12 protein b4232 or its homologue (shown in Table I the 5th or 7 row, the 607th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferred starch and/or Mierocrystalline cellulose be increased in 35% and 61% or more between.

At yeast saccharomyces cerevisiae protein YHR072W-A or its homologue (shown in Table I the 5th or 7 row, the 625th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give in one embodiment fine chemicals, preferably myo-inositol be increased in 27% and 53% or more between.

[0046.0.25.25] in one embodiment, at e. coli k12 protein b0019 or its homologue, for example under the situation that the activity of Na+/H+-exchanger matter nhaA superfamily is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 332nd row) of Na+/H+ antiport protein-active, give each fine chemicals, preferably sucrose and more polyose increase.

In one embodiment, at e. coli k12 protein b0138 or its homologue, for example have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 309th row) of umbrella portion sample adhesin protein active, give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at e. coli k12 protein b0161 or its homologue, for example under the situation that the activity of Helicobacterium serine protease superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of pericentral siphon serine protease (heat shock protein) (for example shown in Table I the 5th or 7 row, the 295th, 303 and 323 row), give each fine chemicals, preferred fructose, glucose and/or raffinose and more polyose increase.

In one embodiment, at e. coli k12 protein b0252 or its homologue, for example have with helicase and ligase enzyme and have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 290th row) of the b0252 protein active of similarity, give each fine chemicals, preferred starch and/or Mierocrystalline cellulose and more polyose increase.

In one embodiment, at e. coli k12 protein b0290 or its homologue, for example under the situation that the activity of intestinal bacteria yagW protein superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of receptor protein (for example shown in Table I the 5th or 7 row, the 310th row), give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at e. coli k12 protein b0695 or its homologue, for example have under the situation that the activity of proteins of sensibility histidine kinase superfamily homology is enhanced, preferably, have under the situation that the activity of sensibility histidine kinase in the reactive two-part regulator control system (for example shown in Table I the 5th or 7 row, the 296th row) is enhanced, give each fine chemicals, preferred fructose and more polyose increase.

In one embodiment, at e. coli k12 protein b0730 or its homologue, for example under the situation that the activity of transcriptional GntR superfamily is enhanced, preferably, have under the activity situation about being enhanced that succinic thiokinase operon transcriptional and acyl reply regulatory gene (for example shown in Table I the 5th or 7 row, the 324th row), give each fine chemicals, preferred raffinose and more polyose increase.

In one embodiment, at e. coli k12 protein b1430 or its homologue, for example under the situation that hemagglutinin hag1 superfamily activity of proteins is enhanced, preferably, have under the activity situation about being enhanced of the S-adenosine of giving the tellurous acid resistance-L-methionine-dependent methyltransgerase (for example shown in Table I the 5th or 7 row, the 291st row) of supposition, give each fine chemicals, preferred starch and/or Mierocrystalline cellulose and more polyose increase.

In one embodiment, at e. coli k12 protein b1693 or its homologue, for example under the situation that the activity of 3-dehydroquinate dehydratase superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of 3-dehydroquinate dehydratase (for example shown in Table I the 5th or 7 row, the 292nd row), give each fine chemicals, preferred starch and/or Mierocrystalline cellulose and more polyose increase.

In one embodiment, at e. coli k12 protein b1701 or its homologue, for example have under the situation that 4-Coumarate-CoA ligase and/or the active activity of proteins of Acetate-CoA ligase superfamily homology be enhanced, preferably, coenzyme A dependency ligase enzyme with Photinus pyralis LUC-sample ATP enzymatic structure territory " under the activity situation about being enhanced of (for example shown in Table I the 5th or 7 row, the 325th row), give each fine chemicals, preferred raffinose and more polyose increase.

In one embodiment, at e. coli k12 protein b1708 or its homologue, for example under the situation that the activity of protein HI1314 superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of lipoprotein (for example shown in Table I the 5th or 7 row, the 297th and 304 row), give each fine chemicals, preferred fructose and/or glucose and more polyose increase.

In one embodiment, at e. coli k12 protein b1886 or its homologue, for example under the situation that the activity of methyl acceptor chemotactic protein matter superfamily is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 326th row) of methyl acceptor chemotactic protein matter II and/or aspartic acid transmitter receptor active, give each fine chemicals, preferred raffinose and more polyose increase.

In one embodiment, at e. coli k12 protein b1926 or its homologue, for example have under the activity situation about being enhanced of the active protein of flagellin matter fliT (for example shown in Table I the 5th or 7 row, the 298th and 305 row), give each fine chemicals, preferred fructose and/or glucose and more polyose increase.

In one embodiment, at e. coli k12 protein b2023 or its homologue, for example have under the situation that the activity of the amide transferase hisH superfamily of trpG homology is enhanced, preferably, have under the activity situation about being enhanced of the active protein of glutamine acid amides transferring enzyme subunit (for example shown in Table I the 5th or 7 row, the 311st row) of imidzoleglycerol phosphate synthase heterodimer, give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at e. coli k12 protein b2597 or its homologue, for example under the situation that the activity of pseudomonas putida putative protein matter rpoX superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of rrna correlation factor (for example shown in Table I the 5th or 7 row, the 299th row), give each fine chemicals, preferred fructose and more polyose increase.

In one embodiment, at e. coli k12 protein b2599 or its homologue, for example have under the situation that the activity of the pheA bifunctional enzyme superfamily of prephenate dehydratase homology is enhanced, preferably, have bifunctional enzyme: under the activity situation about being enhanced of chorismate mutase (N-end) and the active protein of prephenate dehydratase (C-end) (for example shown in Table I the 5th or 7 row, the 306th row), give each fine chemicals, preferred glucose and more polyose increase.

In one embodiment, at e. coli k12 protein b2664 or its homologue, for example under the situation that the activity of transcriptional gabP superfamily is enhanced, preferably, have under the activity situation about being enhanced of " having the transcription repressor (GntR family) of DNA " active protein (for example shown in Table I the 5th or 7 row, the 300th and 327 row) in conjunction with wing spirane structure territory, give each fine chemicals, preferred fructose and/or raffinose and more polyose increase.

In one embodiment, at e. coli k12 protein b2699 or its homologue, for example under the situation that the activity of recombinant protein recA superfamily is enhanced, preferably, have under the situation that the activity of " DNA chain exchange and recombinant protein with proteolytic enzyme and nuclease " active protein (for example shown in Table I the 5th or 7 row, the 312nd and 328 row) is enhanced, give each fine chemicals, preferably myo-inositol and/or raffinose and more polyose increase.

In one embodiment, at e. coli k12 protein b3172 or its homologue, for example under the situation that the activity of arginyl succsinic acid synthase superfamily is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 313rd row) of arginyl succsinic acid synthase activity, give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at e. coli k12 protein b3231 or its homologue, for example under the situation that the activity of intestinal bacteria ribosomal protein L13 superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of 50S ribosomal subunit protein matter L13 (for example shown in Table I the 5th or 7 row, the 293rd row), give each fine chemicals, preferred starch and/or Mierocrystalline cellulose and more polyose increase.

In one embodiment, at e. coli k12 protein b3430 or its homologue, for example under the situation that the activity of Cori ester adenylyl transferase superfamily is enhanced, preferably, have under the activity situation about being enhanced of the active protein of Cori ester adenylyl transferase (for example shown in Table I the 5th or 7 row, the 314th row), give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at e. coli k12 protein b3601 or its homologue, for example have under the situation that the activity of the active protein of transcription repressor that utilizes for N.F,USP MANNITOL (for example shown in Table I the 5th or 7 row, the 329th row) is enhanced, give each fine chemicals, preferred raffinose and more polyose increase.

In one embodiment, at e. coli k12 protein b4129 or its homologue, for example under the situation that the activity of Lysine-tRNA ligase superfamily is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 315th row) of (derivable) Methionin tRNA synthase activity, give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at e. coli k12 protein b4239 or its homologue, for example have under the situation that the activity of the alpha-glucosidase superfamily of α-Dian Fenmei core homology is enhanced, preferably, have under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 301st, 307 and 333 row) of trehalose-6-P hydrolytic enzyme activities, give each fine chemicals, preferred fructose, glucose and/or sucrose and more polyose increase.

In one embodiment, at e. coli k12 protein b4327 or its homologue, for example under the situation that the activity of protein b2409 superfamily is enhanced, preferably, have under the activity situation about being enhanced of " HTH-type transcriptional (LysR family) " active protein (for example shown in Table I the 5th or 7 row, the 302nd row) with periplasmic binding protein matter structural domain, give each fine chemicals, preferred fructose and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR184W or its homologue, for example under the situation that YBR184W protein superfamily activity of proteins is enhanced, preferably, under the activity situation about being enhanced of YBR184W protein (for example shown in Table I the 5th or 7 row, the 330th row), give each fine chemicals, preferred raffinose and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YBR204C or its homologue, for example under the activity situation about being enhanced of peroxysome lipase protein (for example shown in Table I the 5th or 7 row, the 316th row), give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YDR112W or its homologue, for example under the activity situation about being enhanced of YDR112W protein (for example shown in Table I the 5th or 7 row, the 317th row), give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YER174C or its homologue, for example under the situation that the activity of Trx homology superfamily is enhanced, preferably, under the situation that the activity of the reactive gsh dependency of hydrogen peroxide and superoxide radical oxide-reductase protein matter (for example shown in Table I the 5th or 7 row, the 294th row) is enhanced, give each fine chemicals, preferred starch and/or Mierocrystalline cellulose and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YGR261C or its homologue, for example under the situation that the activity of β-adaptin superfamily is enhanced, preferably, under the situation that the activity of clathrin assembling complex body β adaptin composition protein (for example shown in Table I the 5th or 7 row, the 318th and 331 row) is enhanced, give each fine chemicals, preferably myo-inositol and/or raffinose and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YIL150C or its homologue, for example the S phase (DNA synthetic) initial or finish under the situation that the activity of necessary chromobindins (for example shown in Table I the 5th or 7 row, the 319th row) is enhanced, give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL072C or its homologue, for example under the situation that the activity of yeast saccharomyces cerevisiae membranin YJL072c superfamily is enhanced, preferably, chromosomal DNA duplicates under the activity situation about being enhanced of protein subunit matter (for example shown in Table I the 5th or 7 row, the 308th row) of required GINS complex body, give each fine chemicals, preferred glucose and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YJL099W or its homologue, for example participate under the activity situation about being enhanced of protein (for example shown in Table I the 5th or 7 row, the 320th row) of chitin biosynthesizing and/or its adjusting, give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR044W or its homologue, for example under the activity situation about being enhanced of yeast saccharomyces cerevisiae membranin YOR044w superfamily (for example shown in Table I the 5th or 7 row, the 321st row), give each fine chemicals, preferably myo-inositol and more polyose increase.

In one embodiment, at yeast saccharomyces cerevisiae protein YOR350C or its homologue, for example under the activity situation about being enhanced of yeast saccharomyces cerevisiae MNE1 protein superfamily (for example shown in Table I the 5th or 7 row, the 322nd row), give each fine chemicals, preferably myo-inositol and more polyose increase.

At e. coli k12 protein b0050 or its homologue (shown in Table I the 5th or 7 row, the 604th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals, preferred starch and/or Mierocrystalline cellulose and one or more other carbohydrate increases in one embodiment.

At e. coli k12 protein b0124 or its homologue (shown in Table I the 5th or 7 row, the 608th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b0149 or its homologue (shown in Table I the 5th or 7 row, the 609th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b1318 or its homologue (shown in Table I the 5th or 7 row, the 610th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b1463 or its homologue (shown in Table I the 5th or 7 row, the 611st and 617 and 622 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and/or glucose and/or inositol and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b1539 or its homologue (shown in Table I the 5th or 7 row, the 605th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals, preferred starch and/or Mierocrystalline cellulose and one or more other carbohydrate increases in one embodiment.

At e. coli k12 protein b1736 or its homologue (shown in Table I the 5th or 7 row, the 618th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred glucose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b1961 or its homologue (shown in Table I the 5th or 7 row, the 623rd row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferably myo-inositol and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b2491 or its homologue (shown in Table I the 5th or 7 row, the 612nd and 619 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and/or glucose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b3260 or its homologue (shown in Table I the 5th or 7 row, the 613rd row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b3578 or its homologue (shown in Table I the 5th or 7 row, the 614th and 620 and 626 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and/or of glucose and/or raffinose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b3619 or its homologue (shown in Table I the 5th or 7 row, the 615th and 621 row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and/or of glucose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b3919 or its homologue (shown in Table I the 5th or 7 row, the 606th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals, preferred starch and/or Mierocrystalline cellulose and one or more other carbohydrate increases in one embodiment.

At e. coli k12 protein b4074 or its homologue (shown in Table I the 5th or 7 row, the 624th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferably myo-inositol and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b4122 or its homologue (shown in Table I the 5th or 7 row, the 616th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferred fructose and one or more other carbohydrates in one embodiment increases.

At e. coli k12 protein b4232 or its homologue (shown in Table I the 5th or 7 row, the 607th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, give fine chemicals, preferred starch and/or Mierocrystalline cellulose and one or more other carbohydrate increases in one embodiment.

At yeast saccharomyces cerevisiae protein YHR072W-A or its homologue (shown in Table I the 5th or 7 row, the 625th row), for example have such as in [0022.0.25.25] under the active activity of proteins of the definition situation about being enhanced, preferably, giving fine chemicals, preferably myo-inositol and one or more other carbohydrates in one embodiment increases.

[0047.0.0.25] to [0048.0.0.25]: see that [0047.0.0.0] is to [0048.0.0.0]

[0049.0.25.25] has to give and improves starch and/or Mierocrystalline cellulose quantity or the active protein of level and preferably have the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise Table IV the 7th row, the sequence of consensus sequence shown in 290-294 and/or 604-607 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 290-294 and/or 604-607 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 290-294 and/or 604-607 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that starch and/or Mierocrystalline cellulose level increase of giving described in the literary composition.

Have to give and improve fructose quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise Table IV the 7th row, the sequence of consensus sequence shown in 295-302 and/or 608-616 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 295-302 and/or 608-616 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 295-302 and/or 608-616 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the fructose level of giving described in the literary composition increases.

Have to give and improve amount of glucose or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise Table IV the 7th row, the sequence of consensus sequence shown in 303-308 and/or 617-621 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 303-308 and/or 617-621 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 303-308 and/or 617-621 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that glucose level increases of giving described in the literary composition.

Have to give and improve inositol quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise Table IV the 7th row, the sequence of consensus sequence shown in 309-322 and/or 622-625 are capable and/or as Table II the 5th or 7 row, its function homologue shown in 309-322 and/or 622-625 are capable described in polypeptide or the literary composition, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule shown in 309-322 and/or 622-625 are capable or its function homologue as herein described) sequence of coded polypeptide, and have the activity that the inositol level of giving described in the literary composition increases.

Have to give and improve raffinose quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise Table IV the 7th row, the sequence of consensus sequence shown in 323-331 and/or 626 row and/or as Table II the 5th or 7 be listed as, its function homologue described in polypeptide or the literary composition shown in 323-331 and/or 626 row, perhaps (for example be listed as by Table I the 5th or 7 by nucleic acid molecule or the nucleic acid molecule of the present invention that this paper characterized, nucleic acid molecule or its function homologue as herein described shown in 323-331 and/or 626 row) sequence of coded polypeptide, and have the activity that the raffinose level of giving described in the literary composition increases.

Have to give and improve sucrose quantity or the active protein of level preferably has the structure of polypeptide described herein.In special embodiment, polypeptide that the inventive method is used or polypeptide of the present invention comprise Table IV the 7th row, 332-333 capable shown in the sequence of consensus sequence and/or its function homologue described in polypeptide shown in Table II the 5th or 7 row, 332-333 are capable or literary composition or by the sequence of the coded polypeptide of nucleic acid molecule that this paper characterized or nucleic acid molecule of the present invention (for example by Table I the 5th or 7 row, 332-333 capable shown in nucleic acid molecule or its function homologue as herein described), and have the activity that sucrose level increases of giving described in the literary composition.

[0050.0.25.25] for the purposes of the present invention, term " each fine chemicals " also comprises corresponding salt, ester and ether, is preferably had other monose, disaccharides, oligosaccharides or the polysaccharide etherificate of alkyl, alkenyl or alkynyl alcohol.

[0051.0.25.25] is because that use in the methods of the invention and by the coded proteinic biological activity of nucleic acid molecule of the present invention, the composition that contains each fine chemicals (i.e. raising amount free or in conjunction with chemical) be can produce, carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably composition of sucrose for example comprised.Depend on that the inventive method uses biological selection (for example microorganism or plant), can produce the composition or the mixture of multiple each fine chemicals.

[0052.0.0.0] seen in disclosing of [0052.0.0.25] these paragraphs

[0053.0.25.25] in one embodiment, the inventive method comprises one or more the following steps:

(a) stabilizing protein, described protein is given the protein of nucleic acid molecule encoding of the present invention, or polypeptide of the present invention (for example has as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide) express and increase, have the activity of each fine chemicals of raising described in the literary composition;

(b) stable mRNA, described mRNA are given the coded protein of nucleic acid molecule of the present invention and (are for example had as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide) express and improve or mRNA that coding has an active polypeptide of the present invention of each fine chemicals of the raising described in the literary composition expresses and improves;

(c) improve the protein specific activity, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or the of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide) express and improve, perhaps reduce inhibition regulation and control to polypeptide of the present invention;

(d) generation or the endogenous transcription factor of raising mediating protein expression or the expression of manual transcription factor, described protein is given has the active of each fine chemicals of the raising described in the literary composition, by the coded protein of nucleic acid molecule of the present invention, polypeptide perhaps of the present invention (for example has as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide) express and improve;

(e) by add the activity that one or more external source inducible factors come stimulating protein in biological or its part, described protein is given has the active of each fine chemicals of the raising described in the literary composition, protein or of the present invention polypeptide coded by nucleic acid molecule of the present invention (for example have as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide) express and improve;

(f) transgenosis of expression coded protein, described protein is given the activity with each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide) express and improve;

(g) copy number of raising gene, described gene is given nucleic acid molecule and is expressed raising, and described nucleic acid molecule encoding has the active of each fine chemicals of the raising described in the literary composition, (for example have by nucleic acid molecule encoded polypeptide of the present invention or polypeptide of the present invention as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide);

(h) by adding positive Expression element or removing negative Expression element and improve code book invention polypeptide and (for example have as Table II the 3rd row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in protein or its homologue (for example Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in) active polypeptide) and the expression of native gene, for example can use homologous recombination in promotor, to introduce positive controlling element (as plant 35S enhanser) or remove straining element from control region.Can use other genetic modification methods to destroy straining element or enhancing positive element activity.Can in plant, introduce positive element at random by T-DNA or transposon mutagenesis, and identify that wherein positive element is incorporated near the gene of the present invention and makes genetic expression of the present invention so enhanced strain system;

(i) invent the expression of proteinic gene or protein itself or the growth conditions that active mode is regulated and control plant to strengthen code book, for example can cause that heat shock protein(HSP) strengthens culturing micro-organisms or plant under the higher temperature conditions of expressing, this condition can cause that the enhanced fine chemicals produces; And/or

(j) from natural or mutagenesis resource, select to have the biological of extra high protein active of the present invention and it is cultivated and be purpose biology, for example original seed crop.

[0054.0.25.25] preferably, described mRNA is a nucleic acid molecule of the present invention, and/or give the coded protein of nucleic acid molecule of the present invention or have this paper that to mention the protein that active expression of polypeptides improves be polypeptide of the present invention, for example improving coded protein or having as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, (for example Table II the 5th or 7 is listed as for protein shown in 332-333 is capable or its homologue, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide shown in 332-333 is capable) giving each fine chemicals after active expression of polypeptides or the activity increases.

[0055.0.0.25] to [0064.0.0.25]: see that [0055.0.0.0] is to [0064.0.0.0]

[0065.0.0.25]: see [0065.0.0.0]

[0066.0.0.25] to [0067.0.0.25]: see that [0066.0.0.0] is to [0067.0.0.0]

[0068.0.25.25] with not can to carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably the generation of sucrose causes the mode of disadvantageous effect to introduce sudden change.

[0069.0.0.0] seen in disclosing of [0069.0.0.25] these paragraphs

[0070.0.25.25] will be owing to will give a gene or a plurality of gene (nucleic acid construct of mentioning for example) of nucleic acid molecule of the present invention or expression of polypeptides of the present invention, perhaps code book is invented a proteinic gene or a plurality of gene separately or import biological with other assortment of genes, not only can improve biosynthesizing flux towards end product, can also in biology, improve, modify or from the beginning produce favourable, be preferably new metabolite composition, for example contain high level (from the physiology of nutrition angle) preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably favourable carbohydrate composition of sucrose.

[0071.0.25.25] preferably, with composition of the present invention or composition biological or that its part provides also contain higher amount to the nutrition of animal and human's class or healthy have the metabolite that just influencing or low quantity to nutrition of animal and human's class or healthy metabolite with negatively influencing.Similarly, can improve the number or the activity that input or output other required genes of nutrition or metabolite (comprising that cell biological synthesizes required carbohydrate and the precursor thereof of carbohydrate), to improve the concentration of necessity in cell or the corresponding storage compartment or associated precursors, cofactor or intermediate product.Because polypeptide of the present invention or be used for the raising or the active new generation of the polypeptide active of the inventive method, perhaps, can in host living beings such as plant or microorganism, improve output, production and/or the generation efficient of carbohydrate owing to nucleic acid sequence of the present invention and/or to participating in the raising of biosynthetic other generegulation of carbohydrate (activity of for example passing through to improve the activity of the enzyme that synthesizes precursor or participating in one or more genes of carbohydrate decomposition by destruction).

[0072.0.25.25] therefore might produce more favourable compound in the methods of the invention by influencing metabolism.This type of examples for compounds except polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably the sucrose, also have other carbohydrate, preferred carbohydrate.

[0073.0.25.25] therefore, in one embodiment, the method according to this invention relates to and comprises following method:

(a) provide the non-human being, preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant;

(b) improve polypeptide of the present invention or its homologue (for example Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in) active or by the activity of the polypeptide of following nucleic acid molecule encoding of the present invention, each fine chemicals of for example giving in the biology (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) improves;

(c) under the condition that allows each fine chemicals in biological (preferred microorganism, vegetable cell, plant tissue or plant), to produce, cultivate biological (preferred microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant); With

(d) if expectation is reclaimed (randomly separating) by free and/or each fine chemicals of bonded of biology, microorganism, non-human animal, plant or zooblast, plant or animal tissues or phytosynthesis and other optional free and/or bonded carbohydrate.

[0074.0.25.25] biological (particularly being microorganism, non-human animal, plant or zooblast, plant or animal tissues or plant) be growth in the following manner preferably, promptly not only can reclaim (if expectation can separate) free or each fine chemicals of bonded or free or each fine chemicals of bonded, other dissociates and/or in conjunction with carbohydrate but also can choose generations wantonly, reclaim and separate (if desired).

[0075.0.0.25] to [0077.0.0.25]: see that [0075.0.0.0] is to [0077.0.0.0]

[0078.0.25.25] will be such as biological recovery and the separation (if desired) of microorganism or plant, then can with each fine chemicals directly processing enter in food or the animal-feed or other is used for nutritious prod or medicine or makeup purposes, for example according to following disclosing: US 6,669,962: the starch capsules that is used for the delivery of active medicament; US 20050042737: the starch method; US20050054071: the enzyme that is used for starch processing; US 20050091716: new plant and the process that is used to obtain them; U.S. the patent No. 5,096, and 594 and 5,482,631 disclose the method for purifying cyclitol; U.S. the patent No. 4,997, and 489 disclose in water and to soak Pericarppium Armeniacae Amarum to obtain comprising the syrup of fructose, glucose, inositol and sorbyl alcohol; U.S. the patent No. 5,296, and 364 disclose the microbial process that produces inositol; US.4,734,402; US.4,788,065; US 6,465, and 037 and US 6,355,295: relate to soybean food composition based on carbohydrate; US 6,653, and 451; US 20040128713: relate to the soybean plants that has enough low levels raffinoses, stachyose and phytic acid and have enough high-content sucrose and inorganic phosphate in its seed; US 20050008713 discloses plant carbohydrate composition and the nutritional support that is used for dietary supplement ingredient; They in this paper specific reference as a reference.Can or pass through those skilled in the art's method known and that describe hereinafter and handle and processing and fermentation liquid, tunning, plant or plant product as above-mentioned application.

Generation carbohydrate according to the present invention, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably in the method for sucrose, preferably gather in the crops described biology after the culturing step to genetic modified organism (hereinafter being also referred to as genetically modified organism) and from the various carbohydrates of described bioseparation.

Known and be applicable to that the mode of particular organisms gathers in the crops biology with itself.By the microorganism of fermentation culture, for example bacterium, mosses, yeast and fungi or vegetable cell can reclaim in the liquid medium within, for example by centrifugal, decant or filtered and recycled.Plant grown on the solid medium in a manner known way and results correspondingly.

In a manner known way, for example by extracting and (in due course) other chemistry or physical purification process, for example chemistry and/or enzyme liberating, intermediate processing, crystallography, thermal separation method (as rectificating method) or physical separation method (for example chromatography) are separated carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose from the biomass of results.

For these different methods of setting up gradually, product is for containing carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably the composition of sucrose for example contains carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose, also comprise different amounts, advantageously be 0-99% by weight, preferably be lower than 80% by weight, especially preferably by weight 50%, 40%, 30%, 20%, 20%, 10%, 5%, 3%, 2%, 1%, 0.5%, 0.1%, fermented liquid between 0.01% and 0%, the composition of vegetable particle and cellular constituent.

In one embodiment, preferred plant includes but not limited to: sugar beet, sugar-cane, soybean and/or potato.

[0079.0.0.25] to [0084.0.0.25]: see that [0079.0.0.0] is to [0084.0.0.0]

[0085.0.25.25] with regard to nucleotide sequence as mentioned below, contain the nucleic acid construct of nucleotide sequence mentioned in this article or the biology (=genetically modified organism) that transformed with described nucleotide sequence or described nucleic acid construct with regard to, " transgenosis " refers to all constructs by the generation of genetic manipulation method, preferably wherein:

(a) as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in the nucleotide sequence or derivatives thereof, perhaps

(b) with as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the genetic regulatory element that effectively is connected of nucleotide sequence or derivatives thereof, promotor for example, perhaps

(c) (a) and (b)

In its natural genotypic environment, do not exist or modified by the genetic manipulation method.Modification can be for example the substituting of one or more Nucleotide, add, disappearance, be inverted or insert." natural genotypic environment " is meant the natural dyeing body locus in playing eozoan or is present in the genomic library.With regard to genomic library, the natural genotypic environment of nucleotide sequence preferably remains unchanged to small part.Environment and nucleotide sequence link to each other and have the 50bp sequence length at least in a side at least, preferred 500bp at least, especially preferred 1000bp at least, especially especially preferably 5000bp at least.

[0086.0.0.25] to [0087.0.0.25]: see that [0086.0.0.0] is to [0087.0.0.0]

[0088.0.25.25] is in an advantageous embodiment of the present invention, and be biological for because the plant form that expressed nucleic acid molecule of the present invention has advantageously been modified each fine chemicals content.Owing to for example be used for providing the nutritive value of the plant of nutrition to depend on above-mentioned carbohydrate and as total scale of construction of the carbohydrate of food energy derive, so this is very important for the human and animal.

[0088.1.0.25] to [0095.0.0.25]: see that [0088.1.0.0] is to [0095.0.0.0]

[0096.0..25.25] improves nucleotide sequence of the present invention or protein expression and transforming protein matter or polypeptide or compound (as required fine chemicals such as carbohydrate in the biology, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably pond of sucrose) and combines and can be used for producing each fine chemicals in another embodiment preferred of the present invention.

[0097.0.0.0] seen in disclosing of [0097.0.0.25] these paragraphs

[0098.0.25.25] in preferred embodiments, each fine chemicals (carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose) be produce according to the present invention and carry out isolating where necessary.Produce according to the present invention carbohydrate or its mixture or with the mixture of other compound be favourable.

[0099.0.25.25] for microbial fermentation, aforementioned sugars, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose can be accumulated in substratum and/or the cell.If microorganism is used for method of the present invention, the aftertreatment fermented liquid can cultivated.Depend on needs, can from fermented liquid, remove the biomass of some or all, perhaps also biomass can be stayed in the fermented liquid by the combination of separation method such as centrifugal, filtration, decant or these methods.Can reduce or concentrated broth by reverse osmosis or nanofiltration by currently known methods (as rotatory evaporator, thin layer evaporator, falling film evaporator) subsequently.Advantageously add other compound that is used to prepare, for example W-Gum or silicic acid then.Subsequently can be by freeze-drying, spraying drying and spraying choosing grain or the compound of handling spissated fermented liquid and being advantageously used in preparation by additive method.Preferably, in a known way, for example the combination by extracting, distillation, crystallization, chromatogram or these methods from biological as microorganism or plant or biological therein or its substratum of growing, perhaps from biology and substratum, separate each fine chemicals or carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably composition of sucrose.These purification process can use separately, perhaps with aforesaid method as separating and/or concentration method is used in combination.

[0100.0.25.25] comprise the method according to this invention synthetic comprise carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably the transgenic plant of sucrose can advantageously directly be put on market, and do not need institute's synthetic oils, lipid or lipid acid are separated.The plant meaning that is used for the inventive method is meant complete plant and all plant parts, plant organ or plant part, for example leaf, stem, seed, root, stem tuber, flower pesticide, fiber, fruit, root hair, handle, embryo, callus, cotelydons, petiole, results material, plant tissue, breeding tissue and cell culture, it derives from actual transgenic plant and/or can be used to produce transgenic plant.In this context, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryo tissue.

Yet, each fine chemicals that the method according to this invention produces can also be from biology, favourable from plant with they extract (for example aqueous extract) form or as fiber (under starch and/or cellulosic situation), as degraded product (chemistry or zymetology degraded) or crystallized product or as their ether and/or ester and/or free carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose isolated in form.Can obtain each fine chemicals of producing by present method by gathering in the crops biology (from biology therein the growing crop or) from the field.This can be by for example realization is processed or extracted to axis, seed, root, stem tuber, flower pesticide, fiber, fruit to plant part.In order to improve the efficient of extraction, with vegetable material, particularly stem, seed, root, stem tuber, flower pesticide, fiber, fruit clean, softening (temper) and to shell in case of necessity and peel off be favourable.Extract and/or carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose can need not heat cold be beaten or cold-press process obtains by being called.In order more like a cork plant part (particularly stem, seed, root, stem tuber, flower pesticide, fiber, fruit) to be disperseed, can be before with its pulverizing, steaming or roasting.Then, will be carry out pretreated plant part squeezing or with solvent or water extraction in this mode.The subsequent removal solvent.At material is under the situation of microorganism, and the step after the results is for example directly to extract and do not need further procedure of processing, otherwise the several different methods of being familiar with by those skilled in the art after destruction is extracted.After this, resulting product is further processed i.e. degraded, crystallization and/or refining.

[0101.0.0.0] seen in disclosing of [0101.0.25.25] these paragraphs

[0102.0..25.25] carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose can advantageously be analyzed and detect by MS or MSMS method by HPLC or GC separation method.By the standard method of analysis GC that uses many places to describe, GC-MS, LC, LC-MSMS or TLC can clearly detect the biology analysis of recombinating and contain carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably existence of the product of sucrose.Can be by anion-exchange chromatography (Cataldi etc., the Anal Chem. that has the pulse ampere detector; 72 (16): 3902-7,2000), use from R-Biopharm and Roche or from the detection kit of Megazyme (Ireland) and further analyze carbohydrate in the plant milk extract by zymetology " BioAnalysis ".

[0103.0.25.25] in preferred embodiments, the present invention relates to produce the method for each fine chemicals, described method is included in the expression that at least a nucleic acid molecule takes place in biology or its part, or the expression that in biological or its part, produces at least a nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleic acid molecule:

(a) preferably encoding mature form polypeptide or its segmental nucleic acid molecule at least, described polypeptide have as Table II the 5th or 7 row, are respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the 332-333 sequence shown in capable; Described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

(b) preferably comprise the nucleic acid molecule of mature form nucleic acid molecule at least, described mature form nucleic acid molecule has as Table I the 5th or 7 row, is respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the 332-333 sequence shown in capable;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

(d) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(e) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

(f) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) coding by the fragment or the epi-position of one of the nucleotide sequence of (a) to (e) (preferred (a) to (c)) coded polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(h) nucleic acid molecule, its comprise by use have as Table III the 7th row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the primer of sequence nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give the increase of each fine chemicals quantity in biology or its part;

(i) coding by monoclonal antibody from expression library for example isolated polypeptide and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (h) (preferred (a) is to (c));

(j) encoded polypeptides comprise have as Table IV the 7th row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence consensus sequence and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

(k) comprise the nucleic acid molecule of one or more coded polypeptide aminoacid sequences and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity, described polypeptid acid sequence coding Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the structural domain of polypeptide; With

(l) can be by under stringent condition, obtaining and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with the probe (sequence that contains one of the nucleic acid molecule of (a) to (k), preferred (a) to (c)) or the suitable library of screening of fragment (having the 15nt at least of (a) to (k), preferred (a) to (c) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt);

Or described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence.

[00103.1.0.4.] in one embodiment, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I A the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I A the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II A the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in polypeptide of sequence.

[00103.2.0.4.] in one embodiment, nucleic acid molecule of the present invention and Table I B the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I B the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I B the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II B the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in polypeptide of sequence.

[0104.0.25.25] in one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in the sequence difference be one or more Nucleotide.In one embodiment, the used nucleic acid of nucleic acid molecule of the present invention or the inventive method not by Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in sequence form.In one embodiment, nucleic acid molecule of the present invention and Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the identity of sequence less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule do not encode Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in polypeptide of sequence.

[0105.0.0.25] to [0107.0.0.25]: see that [0105.0.0.0] is to [0107.0.0.0]

Advantageously improved in [0108.0.25.25] method of the present invention and had Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleic acid molecule of sequence shown in 332-333 is capable, from Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, aminoacid sequence shown in 332-333 is capable is derived or is listed as from containing Table IV the 7th, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the polypeptide deutero-nucleic acid molecule of consensus sequence shown in 332-333 is capable, perhaps its coding has as Table II the 3rd, 5 or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, enzymic activity of polypeptide shown in 332-333 is capable or bioactive polypeptide or for example to give each fine chemicals behind its expression or active the increasing be carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably derivative or the homologue that increase of sucrose.

[0109.0.0.0] seen in disclosing of [0109.0.0.25] these paragraphs

[0110.0.0.9] helps the inventive method and coding has the polypeptide of polypeptide of the present invention or the used polypeptide active of the used or of the present invention process of the inventive method (for example as Table II the 5th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, protein shown in 332-333 is capable or by as Table I the 5th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the protein of nucleic acid molecule encoding shown in 332-333 is capable or its homologue are (as Table II the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, shown in 332-333 is capable)) nucleic acid molecule can from generally open database, determine.

[0111.0.0.0] seen in disclosing of [0111.0.0.25] these paragraphs

The nucleic acid molecule that uses in [0112.0.25.25] the inventive method is the isolated nucleic acid sequences form, its coding has as Table II the 3rd row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the polypeptide of polypeptide active shown in 332-333 is capable or have as Table II the 5th and 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the polypeptide of peptide sequence shown in 332-333 is capable, and give carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level increases.

[0113.0.0.25] to [0120.0.0.25]: see that [0113.0.0.0] is to [0120.0.0.0]

[0121.0.25.25] yet, can also use artificial sequence, the nucleotide sequence that exists in described artificial sequence and the biology has the difference of one or more bases, or with biology in the peptide sequence that exists the difference of one or more amino acid moleculars is arranged, particularly with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, peptide sequence shown in 332-333 is capable or its functional homologue as herein described have the difference of one or more amino acid moleculars, described artificial sequence is preferably given aforementioned activity, is promptly improving Table II the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, giving of the present invention fine chemicals level after the activity of peptide sequence for the 332-333 of sucrose shown in capable increases.

[0122.0.0.25] to [0127.0.0.259]: see that [0122.0.0.0] is to [0127.0.0.0]

The synthetic oligonucleotide primer thing that [0128.0.9.9] polymerase chain reaction (PCR) amplification is used is (for example as Table III the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, primer shown in 332-333 is capable to) can be based on sequence shown in this paper, for example Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable or from as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the sequence of sequence deutero-shown in 332-333 is capable produces.

[0129.0.25.25] yet, can compare the conservative region of identifying in the multiple biology by carrying out protein sequence, therefrom can draw conservative region and can obtain degenerated primer conversely with the employed polypeptide of process of the present invention (the particularly sequence of polypeptide of the present invention).Conservative region is the zone of changes little in the amino acid of a specific position in several homologues of different sources.Table IV the 7th row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in consensus sequence from described comparison.

[0130.0.25.25] can use then degenerated primer by pcr amplification have aforementioned activity (as improve to express or activity after give carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose increases) the new protein fragment or comprise as described in segmental protein.

[0131.0.0.25] to [0138.0.0.25]: see that [0131.0.0.0] is to [0138.0.0.0]

[0139.0.25.25] can be by other dna sequence encodings from the polypeptide of the aforementioned activity of having of other biological (promptly giving each fine chemicals level increases), described dna sequence dna under loose hybridization conditions with Table I, preferred Table I B the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively, 295-302 and/or 608-616 for fructose are capable, 303-308 and/or 617-621 for glucose are capable, 309-322 and/or 622-625 for inositol are capable, 323-331 and/or 626 row for raffinose, for the 332-333 of sucrose capable shown in sequence hybridization, and the coding expression has each fine chemicals, particularly starch of increasing and/or Mierocrystalline cellulose and/or fructose and/or glucose and/or inositol and/or raffinose and/or the active peptide of sucrose.

[0140.0.0.25] to [0146.0.0.25]: see that [0140.0.0.0] is to [0146.0.0.0]

[0147.0.25.25] in addition, nucleic acid molecule of the present invention comprises one of nucleotide sequence with aforementioned nucleic acid molecule or its part complementary nucleic acid molecule.With Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in one of nucleotide sequence complementary nucleic acid molecule be meant these molecules: it is fully complementary with one of described nucleotide sequence, thereby can hybridize and form stable duplex with one of described nucleotide sequence.Preferably under stringent hybridization condition, hybridize.Yet, the nucleic acid molecule base pairing that the complement of one of open sequence of this paper is preferably known according to the technician and with its complementary sequence.For example, base A and G carry out base pairing with base T and U or C respectively, and vice versa.The modification of base can influence the base pairing mating partner.

[0148.0.25.25] nucleic acid molecule of the present invention contains following nucleotide sequence: with Table I, preferred Table I B the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, nucleotide sequence shown in 332-333 is capable or its portion homologous are at least about 30%, 35%, 40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more preferably at least about 70%, 80% or 90%, even more preferably at least about 95%, 97%, 98%, 99% or higher, and preferably have aforementioned activity, particularly having after the activity of the product that improves its activity or the described sequence gene of encoding or its homologue increases carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably activity of sucrose.

[0149.0.25.25] nucleic acid molecule of the present invention comprises nucleotide sequence, described sequence and Table I, preferred Table I B the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, one of nucleotide sequence shown in 332-333 is capable or the hybridization of its part, preferably in as literary composition, hybridize under the defined stringent condition, and coding has above-mentioned activity and (as gives carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose increases) and randomly as Table II the 5th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the protein of protein active shown in 332-333 is capable.

[00149.1.25.25] randomly, in one embodiment, with Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleotide sequence of one of nucleotide sequence shown in 332-333 is capable hybridization has another or various active, described activity for be listed as 290-294 as Table II the 3rd and/or 604-607 capable, 295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, protein shown in 323-331 and/or 626 row is known activity or is used for these protein of note.

[0150.0.25.25] in addition, nucleic acid molecule of the present invention can only contain Table I, preferred Table I B the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the part of the coding region of one of nucleotide sequence shown in 332-333 is capable, for example can be used as the fragment of the biologically-active moiety of fragment that probe or primer use or code book invention polypeptide or polypeptide that the inventive method is used, promptly have aforementioned activity, for example when its active raising, give carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose increases.The nucleotide sequence of identifying from the clone of protein coding gene of the present invention can produce probe and primer, is used for identifying and/or clone its homologue from other cell types or biology.Probe/primer generally contains the oligonucleotide of basic purifying.Oligonucleotide generally contains one section nucleotides sequence column region, and described nucleotides sequence column region is listed as with Table I the 5th or 7 under stringent condition, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the sense strand of one of sequence shown in 332-333 is capable, Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the antisense sequences of one of sequence shown in 332-333 is capable or its be natural exist mutant at least about 12,15, preferred about 20 or 25, more preferably 40,50 or 75 continuous nucleotide hybridization.Can as shown in embodiment, in the PCR reaction, use the homologue of cloning polypeptide of the present invention or polypeptide that the inventive method is used based on the primer of Nucleotide of the present invention, for example the primer of describing in the embodiment of the invention.With Table III the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the right PCR of primer shown in 332-333 is capable will produce as Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the fragment of polynucleotide sequence shown in 332-333 is capable or its gene product.

[0151.0.0.25]: see [0151.0.0.0]

[0152.0.25.25] nucleic acid molecule encoding polypeptide of the present invention or its part, described polypeptide or its part comprise and are listed as Table II the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the abundant homology of aminoacid sequence shown in 332-333 is capable, make this albumen or its part keep to participate in produce the ability of each fine chemicals, specifically be included in plant or the microorganism as described above or raising carbohydrate as be shown in the examples, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably activity of sucrose level.

[0153.0.25.25] term used herein " fully homology " refers to have protein or its part of following aminoacid sequence, described aminoacid sequence comprises and is listed as Table II the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the amino-acid residue that the aminoacid sequence minimal number was identical or of equal value shown in 332-333 was capable (for example, have to one of peptide sequence of the present invention in the amino-acid residue of the similar side chain of amino-acid residue), produce so that this protein or its part can participate in increasing each fine chemicals.In one embodiment, as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, protein shown in 332-333 is capable or its part have Table II the 3rd row for example as herein described, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the activity of polypeptide shown in 332-333 is capable.

[0154.0.25.25] in one embodiment, nucleic acid molecule of the present invention contains the nucleic acid of a code book invention protein part.This protein with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the complete amino acid sequence homology was at least about 30% shown in 332-333 was capable, 35%, 45% or 50%, preferably at least about 55%, 60%, 65% or 70%, more preferably at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% also most preferably at least about 95%, 97%, 98%, 99% or higher, and have aforementioned activity, for example preferably giving each fine chemicals increases.

[0155.0.0.25] to [0156.0.0.25]: see that [0155.0.0.0] is to [0156.0.0.0]

[0157.0.25] the present invention relates in addition owing to the genetic code degeneracy is different from Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, one of nucleotide sequence shown in 332-333 is capable (with its part) and thereby code book invention polypeptide, particularly have the polypeptide of aforementioned activity (for example giving each fine chemicals increase in the biology), for example comprise as Table IV the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide of sequence shown in 332-333 is capable or as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleic acid molecule of polypeptide shown in 332-333 is capable or its function homologue.Advantageously, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor comprise or have in another embodiment the nucleotide sequence of coded protein, and described protein comprises or has in another embodiment as Table IV the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, consensus sequence shown in 332-333 is capable or as Table II the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the aminoacid sequence of polypeptide shown in 332-333 is capable or its function homologue.Also in another embodiment, nucleic acid molecule of the present invention or the inventive method nucleic acid molecule used therefor coding full length protein, described full length protein with comprise as Table IV the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, consensus sequence shown in 332-333 is capable or as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the basic homology of aminoacid sequence of polypeptide shown in 332-333 is capable or its function homologue.Yet, in preferred embodiments, nucleic acid molecule of the present invention is not by Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I A the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable is formed.Preferably, nucleic acid molecule of the present invention be Table I B the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the functional homologous compound of nucleic acid molecule or identical with it.

[0158.0.0.25] to [0160.0.0.25]: see that [0158.0.0.0] is to [0160.0.0.0]

[0161.0.25.25] therefore, in another embodiment, length of nucleic acid molecule of the present invention is at least 15,20,25 or 30 Nucleotide.Preferably, it under stringent condition with the nucleic acid molecule that comprises the nucleotide sequence of nucleic acid molecule of the present invention or nucleic acid molecule that the inventive method is used (for example comprise Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the nucleic acid molecule of sequence) hybridize.This nucleic acid molecule preferred length is at least 20,30,50,100,250 or polynucleotide more.

[0162.0.0.25]: see [0162.0.0.0]

[0163.0.25.25] preferably, under stringent condition with Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the nucleic acid molecule of the present invention of sequence hybridization corresponding to naturally occurring nucleic acid molecule of the present invention.The nucleic acid molecule of " natural existence " used herein refers to have the RNA or the dna molecular of the nucleotide sequence (natural protein of for example encoding) that occurring in nature exists.Preferably, this nucleic acid molecule encoding has the natural protein of aforementioned activity (for example improving its expression or its active back or improving protein of the present invention or the inventive method is given each fine chemicals increase after using activity of proteins).

[0164.0.0.25]: see [0164.0.0.0]

[0165.0.25.25] for example, can the sequence of nucleic acid molecule of the present invention or the employed nucleic acid molecule of the inventive method (for example as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence) in produces the Nucleotide replacement of the amino acid replacement that causes " nonessential " amino-acid residue place.

[0166.0.0.25] to [0167.0.0.25]: see that [0166.0.0.0] is to [0167.0.0.0]

[0168.0.25.25] therefore the present invention relates to the nucleic acid molecule that coding has the polypeptide of aforementioned activity (for example giving each fine chemicals increase in biology or its part), and described polypeptide contains the variation to the nonessential amino-acid residue of described activity.These amino acid sequence of polypeptide with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the sequence difference that sequence shown in 332-333 is capable is contained, but kept activity as herein described.This nucleic acid molecule can contain the nucleotide sequence of coded polypeptide, wherein polypeptide contains and is listed as Table II the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, aminoacid sequence shown in 332-333 is capable is at least about 50% identical aminoacid sequence, and can participate in increasing the generation of each fine chemicals after improving its activity (for example it is expressed).Preferably, the protein of this nucleic acid molecule encoding with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable is identical at least about 60%, more preferably with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, one of sequence shown in 332-333 is capable is identical at least about 70%, even more preferably with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable is at least about 80%, 90% or 95% homology, and most preferably with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable is at least about 96%, 97%, 98% or 99% is identical.

[0169.0.0.25] to [0172.0.0.25]: see that [0169.0.0.0] is to [0172.0.0.0]

[0173.0.25.25] for example, it is such sequence that the sequence that has 80% homology in nucleic acid level and SEQ ID NO:28606 sequence is interpreted as, and has 80% homology when promptly stating the aforementioned Gap programmed algorithm of parameter setting and the comparison of SEQ ID NO:28606 sequence before use.

[0174.0.0.25]: see [0174.0.0.0]

[0175.0.25.25] for example, the sequence that has 80% homology at protein level and SEQ ID NO:28607 is interpreted as such sequence, has 80% homology when promptly using the aforementioned programmed algorithm of aforementioned parameters setting and the comparison of SEQ ID NO:28607 sequence.

[0176.0.25.25] is by replacing, insert or lack from being listed as Table II the 5th or 7 according to of the present invention, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the functional equivalent that one of polypeptide obtained shown in 332-333 was capable be listed as according to of the present invention as Table II the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, one of polypeptide had at least 30% shown in 332-333 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85%, 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and by with as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide shown in 332-333 is capable has essentially identical character and discerns.

[0177.0.25.25] is by replacing, insert or disappearance from according to of the present invention as Table I, preferred Table I B the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the functional equivalent that nucleotide sequence obtained shown in 332-333 was capable be listed as according to of the present invention as Table II the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, one of polypeptide had at least 30% shown in 332-333 was capable, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%, preferably at least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or 94%, extremely preferably at least 95%, 97%, 98% or 99% homology, and the coding have with as Table II, preferred Table I B the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the polypeptide of the essentially identical character of polypeptide shown in 332-333 is capable.

[0178.0.0.25]: see [0178.0.0.0]

[0179.0.25.25]: can (particularly be listed as by nucleotide sequence to nucleic acid molecule of the present invention as Table I the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, shown in 332-333 is capable) the middle replacement of introducing one or more Nucleotide, add or disappearance, and thereby in coded protein, introduce one or more amino acid and replace, add or disappearance and produce coding as Table II the 5th or 7 is listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleic acid molecule of the homologue of protein sequence shown in 332-333 is capable.Can by standard technique (as site-directed mutagenesis and PCR mediated mutagenesis) to as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 introduce sudden change in the encoding sequence shown in capable.

[0180.0.0.25] to [0183.0.0.25]: see that [0180.0.0.0] is to [0183.0.0.0]

[0184.0.25.25] is employed to have as Table I the 5th or 7 row, preferred in Table I B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleotide sequence homologous compound of sequence shown in 332-333 is capable, perhaps come Table II the 5th or 7 row freely, preferred Table II B the 7th row, 290-294 and/or 604-607 are capable, 295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the homologous compound of the nucleotide sequence of sequence shown in 332-333 is capable also comprise with shown in nucleotide sequence or aforementioned derivative nucleic acids sequence or its homologue, one of derivative or analogue or their part have at least about 30%, 35%, 40%, or 45% homology, preferably at least 50%, 60% or 70%, more preferably at least 90%, 91%, 92%, 93%, 94% or 95%, even more preferably at least 96%, 97%, 98% or 99% or the allelic variant of higher homology.Allelic variant specifically comprise can by from shown in sequence (preferably from as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence or from the deutero-nucleotide sequence) by disappearance, insert or replace the functional variant that Nucleotide obtains, yet intention is advantageously to keep or improves and synthesize proteinic enzymic activity or the biological activity that obtains.

[0185.0.25.25] in one embodiment of the invention, the employed nucleic acid molecule of nucleic acid molecule of the present invention or the inventive method comprise one or more as Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B the 7th row, 290-294 and/or 604-607 are capable, 295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable.In one embodiment, the preferred nucleic acid molecule comprises the least possible, be listed as Table I the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, other Nucleotide that does not show in arbitrary sequence shown in 332-333 is capable.In one embodiment, nucleic acid molecule contains and is less than 500,400,300,200,100,90,80,70,60,50 or 40 other Nucleotide.In another embodiment, nucleic acid molecule contains and is less than 30,20 or 10 other Nucleotide.In one embodiment, the employed nucleic acid molecule of the inventive method with as Table I the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable is identical.

The employed one or more nucleic acid molecule encodings of [0186.0.25.25] also preferred the inventive method comprise as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide of sequence shown in 332-333 is capable.In one embodiment, nucleic acid molecule encoding is less than 150,130,100,80,60,50,40 or 30 other amino acid.In another embodiment, encoded polypeptides contains and is less than 20,15,10,9,8,7,6 or 5 other amino acid.In one embodiment, the encoded polypeptide that is used for the inventive method with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, resp., preferred in Table II B 290-294 and/or 604-607 are capable, 295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence was identical shown in 332-333 was capable.

[0187.0.25.25] in one embodiment, the employed nucleic acid molecule encoding of nucleic acid molecule of the present invention or present method comprise as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide of sequence shown in 332-333 is capable also contains and is less than 100 other Nucleotide.In another embodiment, described nucleic acid molecule contains and is less than 30 other Nucleotide.In one embodiment, employed nucleic acid molecule is listed as Table II the 5th or 7 with coding in the method, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B 290-294 and/or 604-607 are capable, 295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the encoding sequence of polypeptide was identical shown in 332-333 was capable.

[0188.0.25.25] still has the polypeptide (=protein) of giving the basic biologic activity of polypeptide of the present invention that each fine chemicals increases or the enzyme activity (be its active not have substantially reduction) is to have at least 10% or 20% of wild-type biology activity or enzymic activity, preferred 30% or 40%, preferred especially 50% or 60%, extremely preferably have 80% or 90% or higher polypeptide, advantageously, active with as Table II the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II the 3rd and 5 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, shown in 332-333 is capable and under the same conditions the polypeptide expressed activity is compared and is not reduced substantially.

In one embodiment, polypeptide of the present invention be comprise Table II B the 7th row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence or by its homologue of forming.

[0189.0.25.25] is as Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the homologous compound of sequence shown in 332-333 is capable, or deutero-such as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the homologue of the sequence shown in 332-333 is capable also refers to truncated sequence, cDNA, the single stranded DNA or the RNA of coding and noncoding DNA sequence.The homologue of described sequence will also be understood that to referring to contain the derivative of non-coding region (as UTR, terminator, enhanser or promoter variants).Can replace, insert by one or more Nucleotide and/or disappearance is modified the upstream promoter of described nucleotide sequence, and not disturb promotor, opening code-reading frame (=functional or active ORF) or away from the 3 ' regulatory region (as terminator or other 3 ' regulatory regions) of ORF.Can also improve the activity of promotor by its sequence of modification, or it be replaced fully with active higher promotor (or even promotor of allos biology).Suitable promotor is that those skilled in the art are well-known, and has mentioned hereinafter.

[0190.0.0.0] seen in disclosing of [0190.0.0.25] these paragraphs

[0191.0.0.0] seen in disclosing of [0191.0.0.25] these paragraphs:

Disclosing of [0192.0.0.25] to [0203.0.0.25] these paragraphs sees that [0192.0.0.0] is to [0203.0.0.0]

[0204.0.25.25] therefore in one embodiment, the present invention relates to contain the nucleic acid molecule that is selected from following nucleic acid molecule:

(a) coding, optimized encoding is being listed as Table II the 5th or 7 of mature form at least, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide shown in 332-333 is capable or its segmental nucleic acid molecule, described nucleic acid molecule is given each fine chemicals in biological or its part, i.e. carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases;

(b) comprise, preferably comprise being listed as of mature form at least as Table I the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, nucleic acid molecule shown in 332-333 is capable or its segmental nucleic acid molecule, described nucleic acid molecule are given the increase of each fine chemicals quantity in biological or its part;

(c) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases;

(d) encoded polypeptide with (a) have at least 50% identity and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases to the coded amino acid sequence of polypeptide of the nucleic acid molecule of (c);

(e) under stringent hybridization condition with (a) give the nucleic acid molecule that each fine chemicals quantity increases to the making nucleic acid molecular hybridization of (c) and in biological or its part;

(f) coded polypeptide and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide obtains by substituting, lack and/or add one or more amino acid in the coded amino acid sequence of polypeptide of nucleic acid molecule of (a) to (d) (being preferably (a) to (c));

(g) fragment of coded polypeptide or epi-position and in biological or its part, give the nucleic acid molecule that each fine chemicals quantity increases, described polypeptide is by one of the nucleotide sequence of (a) to (e) (preferred (a) is to (c)) coding;

(h) nucleic acid molecule, it comprises by using as Table III the 5th or 7 row, 290-294 and/or 604-607 are capable, 295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleic acid molecule that primer shown in 332-333 is capable or primer obtain amplification from cDNA library or genomic library, and in biological or its part, give each fine chemicals, i.e. carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably increase of sucrose quantity;

(i) coding is by monoclonal antibody institute's isolated polypeptide and give the nucleic acid molecule that each fine chemicals quantity increases in biological or its part from expression library for example, and wherein said monoclonal antibody is at the nucleic acid molecule encoded polypeptide of one of (a) to (g) (preferred (a) is to (c));

(j) encoded polypeptides contains just like Table IV the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, consensus sequence shown in 332-333 is capable is also given each fine chemicals in biological or its part, i.e. carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity;

(k) coded polypeptide aminoacid sequence and in biological or its part, give each fine chemicals, it is carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity, described peptide coding such as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the structural domain of the polypeptide shown in 332-333 is capable; With

(l) can be by under stringent hybridization condition, screening that suitable nucleic acid library obtains and in biological or its part, giving the nucleic acid molecule that each fine chemicals quantity increases with probe or fragment, described probe contains the sequence of one of (a) to (k) nucleic acid molecule, described fragment has (a) to (h) institute's characterisation of nucleic acids molecule or is listed as Table I the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, nucleic acid molecule shown in 332-333 is capable or coding (optimized encoding is mature form at least) are as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table II B the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, at least the 15nt of the nucleic acid molecule of polypeptide shown in 332-333 is capable, preferred 20nt, 30nt, 50nt, 100nt, the fragment of 200nt or 500nt;

Perhaps described nucleic acid contains and above-mentioned nucleic acid molecule complementary sequence; Thereby the nucleic acid molecule of preferred (a) to (l) is different from as Table I A the 5th or 7 row, is respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the 332-333 sequence shown in capable by one or more Nucleotide.In one embodiment, nucleic acid molecule of the present invention not by Table I A or IB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the 332-333 sequence shown in capable is formed.In one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule not coding schedule IIA or IIB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in polypeptide of sequence.In another embodiment, nucleic acid molecule of the present invention and Table I A or IB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence at least 30%, 40%, 50% or 60% identical and identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In another embodiment, nucleic acid molecule is not encoded as Table II A or IIB the 5th or 7 row, is respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the 332-333 peptide sequence shown in capable.Therefore, in one embodiment, nucleic acid molecule and Table I A or IB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in nucleic acid molecule at least one or a plurality of residue different.Therefore, in one embodiment, nucleic acid molecule encoding of the present invention and Table II A or IIB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the 332-333 polypeptide shown in capable at least one or the different polypeptide of a plurality of amino acid.In another embodiment, Table I A or IB the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, nucleic acid molecule shown in 332-333 is capable is coding schedule IIA or IIB the 5th or 7 row not, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, protein sequence shown in 332-333 is capable.Therefore, in one embodiment, (a) to the coded protein of the nucleotide sequence of (l) not by as Table II A or IIB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence form.In another embodiment, protein of the present invention and Table II A or IIB the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, protein sequence at least 30% shown in 332-333 is capable, 40%, 50% or 60% identical and with Table I A or IIB the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence was less than 100% shown in 332-333 was capable, preferably less than 99.999%, 99.99% or 99.9%, be more preferably less than 99%, 98%, 97%, 96% or 95% is identical.

Disclosing of [0205.0.0.25] to [0206.0.0.25] these paragraphs sees that [0205.0.0.0] is to [0206.0.0.0]

[0207.0.25.25] is as described herein, and nucleic acid construct can also contain other genes of introducing in biology or the cell.Regulatory gene (as inductor, repressor or enzyme gene) introduced host living beings and express therein is possible and favourable, described enzyme is owing to its enzymic activity is regulated one or more genes in the biosynthetic pathway.These genes can be allos source or homology source.In addition, can be favourable have an other biological synthetic gene, perhaps these genes can be positioned on one or more other nucleic acid constructs.The gene that advantageously uses as biosynthesis gene is carbohydrate or starch metabolism, glucose metabolism, carbohydrate metabolism, the metabolic gene of glycolysis-or its combination.As described herein, regulate the sequence or the factor and can be preferably the genetic expression of introducing gene be had positive interaction, thereby make its raising.Therefore, can transcribe signal (as promotor and/or enhanser) by force advantageously at transcriptional level enhancing regulatory element by using.Yet, can also strengthen translation by for example improving mRNA stability or inserting the translation enhancement sequences in addition.

[0208.0.0.25] to [0226.0.0.25]: see that [0208.0.0.0] is to [0226.0.0.0]

[0227.0.25.25]: aforementioned nucleic acid molecule and other assortment of genes can be cloned in nucleic acid construct into of the present invention or the carrier together, perhaps by in host cell (advantageously being vegetable cell or microorganism), transforming some nucleic acid constructs or the different gene of carrier (comprising plasmid) introducing.

Except Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the capable sequence or derivatives thereof of mentioning of 332-333, can advantageously additionally express and/or other genes that suddenly change in biology.Particularly advantageously, extra other genes of expressing at least one carbohydrate biosynthetic pathway in biological (as plant or microorganism).Can also advantageously modify the regulation and control of natural gene, so that gene and/or its gene product no longer place under the regulation mechanism that biology exists.This causes increases synthetic required carbohydrate, and for example, feedback regulation no longer exists or do not exist fully with same degree.In addition can also be advantageously with Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in one or more sequences with generally support or the assortment of genes of intensifier target biological growth or output, for example cause the gene or the generation of microorganism faster growing rate to be coerced, the gene of pathogenic agent or herbicide resistant plants.

[0228.0.25.25]: therefore, in another embodiment of the present invention, cultivated simultaneously and to have crossed expression coding and sugar metabolism, particularly polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably at least one nucleic acid of sucrose synthesized related protein or the biology of a gene.

[0229.0.25.25]: can make up the sequence of other favourable nucleotide sequences of expression with sequence that present method is used and/or aforementioned biosynthesis gene for other gene of coding carbohydrate biosynthetic pathway.These genes cause carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose increases.

[0230.0.0.0] seen in disclosing of [0230.0.0.25] these paragraphs

[0231.0.25.25] in another advantageous embodiment of the inventive method, the employed biology of present method is the protein that has weakened the degraded carbohydrate simultaneously, especially passes through those biologies of the expression rate of reduction corresponding gene.

[0232.0.0.25] to [0276.0.0.25]: see that [0232.0.0.0] is to [0276.0.0.0]

[0277.0..25.25] can separate each fine chemicals that produces by the method that the technician is familiar with from biology.For example by extraction, chemistry or zymetology degraded, crystallization, salt precipitation and/or different chromatographic process etc.Method of the present invention can be criticized formula, half batch of formula or carry out continuously.Each fine chemicals of producing by this process can be by obtaining from crop (biology is grown therein) or field results biology.This can be by squeezing plant part or extract realization.

[0278.0.0.25] to [0282.0.0.25]: see that [0278.0.0.0] is to [0282.0.0.0]

[0283.0.25.25]: in addition, can from cell (for example endotheliocyte), separate and give the natural polypeptides that each fine chemicals increases in biology or its part, for example use antibody of the present invention as mentioned below, for example anti-as Table II the 3rd row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, protein shown in 332-333 is capable, perhaps as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the antibody of polypeptide or its antigen part shown in 332-333 is capable, it can utilize polypeptide of the present invention or its fragment (being polypeptide of the present invention) to produce by standard technique.Monoclonal antibody preferably.

[0284.0.0.25]: see [0284.0.0.0]

[0285.0.25.25] the present invention relates to have as Table II the 5th or 7 row in one embodiment, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable or by as Table I the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the coded polypeptide of sequence of nucleic acid molecule shown in 332-333 is capable or its function homologue.

[0286.0.25.25] in an advantageous embodiment, improved in the inventive method contain just like Table IV the 7th row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in consensus sequence or by the activity of its polypeptide of forming.In another embodiment, the present invention relates to contain just like Table IV the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, consensus sequence shown in 332-333 is capable or by its polypeptide of forming, wherein 20 or still less, preferred 15 or 10, preferred 9,8,7 or 6, more preferably 5 or 4, even more preferably 3, even more preferably 2, even more preferably 1, most preferably 0 specified amino acid position can be by arbitrary aminoacid replacement and/or disappearance.In one embodiment, the present invention relates to comprise the inventive method of polypeptide or relate to comprise more than one as Table IV the 7th row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the polypeptide of consensus sequence (each row).

[0287.0.0.25] to [0290.0.0.25]: see that [0287.0.0.0] is to [0290.0.0.0]

[0291.0.25.25] in an advantageous embodiment, method of the present invention comprises improving and contains plant or microorganism specificity consensus sequence or by its polypeptide of forming.Therefore, in one embodiment, the present invention relates to comprise plant or microorganism specificity consensus sequence or by its polypeptide of forming.

In one embodiment, described polypeptide of the present invention is different from as Table II A or IIB the 5th or 7 row, is respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, the 332-333 sequence shown in capable by one or more amino acid.In one embodiment, polypeptide passes through more than 1,2,3,4,5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even be more preferably more than 40,50 or 60 amino acid are different from as Table II A or IIB the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable, and, preferably, the sequence of polypeptide of the present invention is passed through 80% or 70% amino acid at the most, preferably at the most 60% or 50%, be more preferably at the most 40% or 30%, even be more preferably at the most that 20% or 10% amino acid is different from as Table II A or IIB the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable.In another embodiment, described polypeptide of the present invention can't help as table Table II A or IIB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence form.

[0292.0.0.25]: see [0292.0.0.0]

[0293.0.25.25]: in one embodiment, the present invention relates to give in biology or its part that each fine chemicals increases and by nucleic acid molecule of the present invention or process nucleic acid molecule used therefor encoded polypeptides of the present invention.In one embodiment, polypeptide of the present invention have by one or more amino acid with as Table II A or IIB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in other sequence of sequence phase region.In another embodiment, polypeptide of the present invention can't help as Table II A or IIB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence form.In another embodiment, polypeptide of the present invention is identical less than 100%, 99.999%, 99.99%, 99.9% or 99%.In one embodiment, described polypeptide not by Table I A or IB the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the coded sequence of nucleic acid molecule form.

[0294.0.25.25] in one embodiment, the present invention relates to have as Table II the 3rd row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the polypeptide of activity of proteins shown in 332-333 is capable, it is by one or more amino acid, preferably more than 5,6,7,8 or 9 amino acid, preferably pass through more than 10,15,20,25 or 30 amino acid, even more preferably more than 40,50 or 60 amino acid, but even more preferably by being less than 70% amino acid, more preferably by being less than 50%, even more preferably less than 30% or 25%, more preferably less than 20% or 15%, even be different from more preferably less than 10% as Table II A or IIB the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, sequence shown in 332-333 is capable.

[0295.0.0.25] to [0297.0.0.25]: see that [0295.0.0.0] is to [0297.0.0.0]

The chemical of [0297.1.25.25] non-polypeptide of the present invention be for example do not have Table II the 3rd, 5 or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the active polypeptide of polypeptide.

[0298.0.25.25]: polypeptide of the present invention can participate in method of the present invention.Polypeptide or its part preferably comprise as with Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in the abundant homologous aminoacid sequence of aminoacid sequence.Protein portion is preferably biologically-active moiety as herein described.Preferably, polypeptide that the inventive method is used have with as Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in the identical aminoacid sequence of sequence.

[0299.0.25.25]: in addition, this polypeptide can have by the nucleotide sequence coded aminoacid sequence of following character, the nucleotide sequence hybridization of this nucleotide sequence and nucleic acid molecule of the present invention (preferably hybridizing under above-mentioned stringent condition).Therefore, this polypeptide has by following nucleotide sequence coded aminoacid sequence, described nucleotide sequence with as Table I the 5th or 7 be listed as, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the homology of one of nucleotide sequence sequence shown in 332-333 is capable is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, even more preferably at least about 96%, 97%, 98%, 99% or higher.The preferred polypeptide of the present invention preferably has at least a activity of the present invention and activity as herein described.As described above, the preferred polypeptide of the present invention comprise with as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the nucleotide sequence or the coded aminoacid sequence of its homologue of nucleotide sequence hybridization (preferred hybridize under stringent condition).

[0300.0.25.25]: therefore, described in detail as this paper, polypeptide of the present invention since natural variation or mutagenesis can be on aminoacid sequence with as Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 capable, 332-333 capable shown in sequence different.Therefore, this polypeptide contains and is listed as Table II A or IIB the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the complete amino acid sequence homology of sequence shown in 332-333 is capable is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or 90%, and more preferably at least about 91%, 92%, 93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%, 99% or higher aminoacid sequence.

[0301.0.0.25]see[0301.0.0.0]

The biologically-active moiety of [0302.0.25.25] polypeptide of the present invention comprises the peptide that contains following aminoacid sequence, described aminoacid sequence is from polypeptide of the present invention or amino acid sequence of polypeptide that the inventive method is used, for example as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the aminoacid sequence of the aminoacid sequence shown in 332-333 is capable or its homologous protein, the amino acid that described peptide comprises is less than the full length protein of the polypeptide of the present invention of total length or polypeptide that the inventive method is used or polypeptide as herein described and of the present invention or homologous peptide that the inventive method is used, and shows the activity of at least a polypeptide of the present invention or polypeptide that the inventive method is used.

[0303.0.0.25]: see [0303.0.0.0]

[0304.0.25.25]: operate nucleic acid molecule of the present invention may cause generation have basically as Table II the 3rd row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in polypeptide active and with as described in the protein that on sequence, there are differences of wild-type protein.These proteinic effects or activity may be enhanced, these are proteinic exist number may than usually the number that exists in the cell more many or for wild-type protein these proteinic effects or activity may be lowered.

[0305.0.0.25] to [0308.0.0.25]: see that [0305.0.0.0] is to [0308.0.0.0]

[0309.0.25.25]: in one embodiment, " protein of the present invention (=the polypeptide) " mentioned or as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, protein shown in 332-333 is capable is meant the polypeptide with corresponding polypeptide of the present invention or the used amino acid sequence of polypeptide of the inventive method, be not listed as and be shown in Table II the 5th or 7, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the polypeptide that " non-polypeptide of the present invention " during 332-333 is capable or " other polypeptide " are meant the aminoacid sequence with corresponding following proteins, described protein and polypeptide of the present invention be homology not basically, preferably with Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide shown in 332-333 is capable is homology not basically, for example do not give activity described in the literary composition or note for or be known as Table II the 3rd row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, it is proteinic and shown in 332-333 is capable from the protein of identical or different biology.In one embodiment, be not shown in Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, " non-polypeptide of the present invention " or " other polypeptide " that 332-333 is capable do not give that each fine chemicals increases in biology or its part.

[0310.0.0.25] to [0334.0.0.25]: see that [0310.0.0.0] is to [0334.0.0.0]

[0335.0.25.25]: confirmed the dsRNAi method to reduce as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, the expression of 323-331 and/or 626 row, the 332-333 nucleotide sequence shown in capable and/or its homologue especially effectively and favourable.Special described in WO 99/32619, the dsRNAi method obviously is better than traditional antisense method.Therefore the present invention also relates in introducing biology, in the preferred introduced plant time (or be derived from its cell, tissue, organ or seed), by reduce as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the expression of nucleotide sequence and/or its homologue cause the double stranded rna molecule (dsRNA molecule) of metabolic activity change.Be used for reducing as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the double stranded rna molecule of the coded protein expression of the nucleotide sequence of sequence or its homologue, article two, one of RNA chain is with basic identical to the small part nucleotide sequence, and another RNA chain and nucleotide sequence is basic identical to the small part complementary strand.

[0336.0.0.25] to [0342.0.0.25]: see that [0336.0.0.0] is to [0342.0.0.0]

[0343.0.25.25]: as describing, in order to cause effective reduction of expression, dsRNA and as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown between the genetic transcription thing of nucleotide sequence or its homologue and nonessential 100% sequence identity.Therefore, advantageously this method can be born the ordering bias that exists as the result of genetic mutation, polymorphism or evolutionary divergence.Therefore, for example from a kind of organism as Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in sequence or its homologue dsRNA that begins to produce, can be used for suppressing expressing accordingly in another organism.

[0344.0.0.25] to[0361.0.0.25]: see that [0344.0.0.0] is to [0361.0.0.0]

[0362.0.25.25]: this, the any nucleic acid that the present invention relates to be used for characterized (for example giving cell or biological or each fine chemicals increase of its part) as the present invention's part carries out genetically modified any cell, described nucleic acid nucleic acid molecule for example of the present invention, nucleic acid construct of the present invention, antisense molecule of the present invention, carrier of the present invention or code book invention polypeptide are (for example capable as Table II 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide shown in 332-333 is capable, for example coding has protein such as Table II the 3rd row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the polypeptide of polypeptide active shown in 332-333 is capable) nucleic acid molecule.Because above-mentioned activity, each fine chemicals content in cell or the biology improves.For example, because regulation and control or manipulation, the cytoactive of polypeptide of the present invention or Nucleotide of the present invention improves, and for example expression or the specific activity owing to research object of the present invention in cell or biological or its part improves.Have as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the transgenosis of the polypeptide of polypeptide active shown in 332-333 is capable is meant in the text because genomic regulation and control or manipulation, in cell or biological or its part, be noted as Table II the 3rd row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide shown in 332-333 is capable (for example has as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, polypeptide of sequence shown in 332-333 is capable) activity is enhanced.Example and the inventive method are described in above.

[0363.0.0.25]: see [0363.0.0.0]

[0364.0.0.25]: see [0364.0.0.0]

[0365.0.0.25] to [0373.0.0.25]: see that [0365.0.0.0] is to [0373.0.0.0]

[0374.0.25.25]: containing the transgenic plant of synthesizing each fine chemicals in the methods of the invention can directly introduce to the market, and does not need to separate institute's synthetic compound.In the methods of the invention, plant is interpreted as all plant parts, plant organ, for example leaf, stem, root, stem tuber or seed or reproductive material or results material or whole plant.In this article, seed comprises all parts of seed, for example plants skin, epidermic cell, seed cell, endosperm or embryonic tissue.Yet, the carbohydrate that produces in the inventive method, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose also can be from plant with free or be bonded to the isolated in form of compound or part.The carbohydrate that produces by this method, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose can by from the culture of biological growth or the field collection of biological collect.This can realize by expression, grinding and/or extracting, salt precipitation and/or ion-exchange chromatography or other chromatographic process of plant part (preferred plant seed, fruit, plant tuber etc.).

[0375.0.0.25] to [0376.0.0.25]: see that [0375.0.0.0] is to [0376.0.0.0]

[0377.0.25.25]: therefore, the invention still further relates to the method according to this invention, separated thus produced comprise carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably composition of sucrose or each fine chemicals that is produced.

[0378.0.25.25]: can separate by this way and be higher than 50% by weight, favourable is higher than 60%, preferably be higher than 70%, especially preferably be higher than 80%, extremely preferably be higher than 90% the carbohydrate that produces in the method, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose.As required, subsequently can with resultant carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose is further purified, then mix if desired, and if suitable the preparation with other activeconstituentss (as VITAMIN, amino acid, carbohydrate, microbiotic etc.).

[0379.0.25.25]: in one embodiment, carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose is the mixture that comprises one or more each fine chemicals.In one embodiment, each fine chemicals here looks like and is meant carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose.In one embodiment, carbohydrate here looks like and is meant the mixture of each fine chemicals.

[0380.0.25.25]: the carbohydrate that obtains by the inventive method, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably suitable synthetic parent material of sucrose as other valuable product.For example, they can combinations with one another or use separately and produce medicine, food, animal-feed or makeup.Therefore, the present invention relates to produce the method for medicine, food, animal-feed, nutritious prod or makeup, described method comprises the step of the inventive method, it comprises that separation produces comprises carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably composition or each fine chemicals (if expectation) of sucrose, but and with pharmaceutically acceptable carrier formulated product or product is mixed with the form of agricultural application.Another embodiment of the present invention is the carbohydrate that produces of the inventive method, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose or the purposes of genetically modified organism in animal-feed, food, medicine, foodstuff additive, makeup or medicine.

[0381.0.0.25] to [0382.0.0.25]: see that [0381.0.0.0] is to [0382.0.0.0]

[0383.0.9.25]：./.

[0384.0.0.25]: see [0384.0.0.0]

[0385.0.25.25]: the fermented liquid that obtains with this kind approach, particularly contain and other compound, particularly with other carbohydrate or constitute the lipid acid blended carbohydrate of lipid, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably fermented liquid of sucrose, the fermented liquid that perhaps comprises microorganism or microorganism part (for example plastid), perhaps comprise carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably fermented liquid of sucrose, dry matter content accounts for 7.5 to 25% of weight usually.Can pass through currently known methods then, for example under rotary evaporator, thin-film evaporator, falling film evaporator help by reverse osmosis or nanofiltration with fermented liquid thickening or concentrated.Then by lyophilize, spraying drying, spraying granulation or by the additive method concentrated broth.

[0386.0..0.25]-/-

[0387.0.0.25] to [0392.0.0.25]: see that [0387.0.0.0] is to [0392.0.0.0]

[0393.0.25.25]: in one embodiment, the present invention relates to identify the method for giving the gene product that each compound generation that becomes more meticulous increases in the cell, it comprises following step:

(a) sample that will comprise candidate gene (be coded in and give the gene product that each fine chemicals increases after the expression) for example the nucleic acid molecule of cell, tissue, plant or microorganism or nucleic acid library contact (for example hybridizing) with nucleic acid molecule of the present invention;

(b) evaluation particularly is listed as Table I the 5th or 7 with nucleic acid molecule of the present invention, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, 332-333 is capable, preferred Table I B 290-294 and/or 604-607 are capable, 295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleic acid molecule that nucleic acid molecule shown in 332-333 is capable is hybridized under lax stringent condition, and randomly separate full length cDNA clone or complete genome group clone;

(c) the candidate nucleic acid molecule is introduced the host cell that is fit to produce each fine chemicals, preferred plant cell or microorganism;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) level of each fine chemicals in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare that each fine chemicals level increase in the host cell is given in its expression with wild-type.

[0394.0.0.25] to [0399.0.0.25]: see that [0394.0.0.0] is to [0399.0.0.0]

[00399.1.9.9]: it is contemplated that by for example seeking the resistance of blocking each fine chemicals synthetic medicine and observing this effect whether depend on as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, active or the expression of the polypeptide shown in 332-333 is capable or its homologue, each fine chemicals that screens raising produces, for example with after the drug incubation with have low and high as Table II the 5th or 7 row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the phenotype more much at one of the biology of activity of proteins shown in 332-333 is capable.

[0400.0.0.25] to [0416.0.0.25]: see that [0400.0.0.0] is to [0416.0.0.0]

[0417.0.9.9]: nucleic acid molecule of the present invention, carrier of the present invention or nucleic acid construct of the present invention can also be used to produce to carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably the inhibitor of sucrose biosynthetic pathway has the biology of resistance.Particularly, expressing excessively of polypeptide of the present invention may make biology, and for example microorganism or plant are anti-can block described biological carbohydrate, particularly each fine chemicals synthetic inhibitor.The biological example as carbohydrate synthetic inhibitor or weedicide in microorganism or the plant of blocking-up is a Mierocrystalline cellulose synthetic inhibitor 2,6-dichlorobenzonitrile (DCB), N2-(1-ethyl-3-hydrocinnamyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2, the 4-hydrazine, it is known as AE F150944 or isoxaben.

[0418.0.0.25] to [0423.0.0.25]: see that [0418.0.0.0] is to [0423.0.0.0]

[0424.0.25.25]: therefore, nucleic acid of the present invention, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, the agonist of identifying with the inventive method, the nucleic acid molecule of identifying with the inventive method can be used to produce each fine chemicals or produce each fine chemicals and one or more other carbohydrates.Therefore, nucleic acid of the present invention or the nucleic acid molecule of identifying with the inventive method or its complementary sequence, polypeptide of the present invention, nucleic acid construct of the present invention, biology of the present invention, host cell, microorganism, plant, plant tissue, vegetable cell or its part, carrier of the present invention, antagonist, antibody of the present invention, the antisense molecule of the present invention identified with the inventive method can be used for reducing each fine chemicals of biological or its part (as cell).

[0425.0.0.25] to [0454.0.0.25]: see that [0425.0.0.0] is to [0453.0.0.0]

[0454.0.25.25] embodiment 8: the influence that analyzing nucleic acid molecules produces fine chemicals

[0455.0.25.25] can be by cultivating down the microorganism of modifying and analyzing substratum and/or raising that the amino acid of cellular component produces is measured in Corynebacterium glutamicum or other microorganism (bacterial strain of producing especially for carbohydrate) genetic modification to the influence of purpose compound (for example glycerine and/or glycerol-3-phosphate) generation at felicity condition (as indicated above).This type of analytical technology is that the technician is well-known, and comprise that spectroscopy, thin-layer chromatography, polytype dyeing process, enzyme and microbial process and analysis mode chromatogram (as high performance liquid chromatography) (consult as Ullman, " Encyclopedia of IndustrialChemistry ", the A2 volume, 89-90 and 443-613 page or leaf, VCH:Weinheim (1985); Fallon, A. waits " Applications of HPLC in Biochemistry " in (1987) " Laboratory Techniques in Biochemistry and MolecularBiology " 17 volumes; Rehm etc. (1993) " Biotechnology ", the 3rd volume, III chapter: " Product recovery andpurification ", 469-714 page or leaf, VCH:Weinheim; Belter, P.A. etc. (1988) " Bioseparations:downstream processing for Biotechnology ", JohnWiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) " Recoveryprocesses for biological Materials ", John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) " Ullmann ' s Encyclopedia of Industrial Chemistry " VCH:Weinheim, B3 volume; 11 chapters, " the Biochemical Separations " of 1-27 page or leaf; And Dechow, F.J. (1989) " Separation and purification techniques inbiotechnology ", Noyes Publications).

[0456.0.0.25]: see [0456.0.0.0]

[0457.0.25.25]: embodiment 9: carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably purifying of sucrose

[0458.0.25.25]: abbreviation: GC-MS, gas-liquid chromatograph/mass spectrum; TLC, thin-layer chromatography.

By use described standard method of analysis GC, GC-MS or TLC to the biology analysis of recombinating can clearly detect carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose exists (1997, Advances on Lipid Methodology, the 4th edition: Christie, OilyPress, Dundee, 119-169; 1998, Gaschromatographie-Massenspektrometrie-Verfahren[Gas chromatography/mass spectrometric methods], Lipide33:343-353).

Can analyze the total carbohydrate that in used biology of the inventive method such as yeast, produces by following process:

By ultrasonic, grinding in the glass pulverizing mill, liquid nitrogen and grinding or by other applicable method with material to be analyzed, as yeast, intestinal bacteria or plant fragmentation.

By in pestle and mortar, pulverizing vegetable material machinery homogenate is extracted so that be more suitable at first.

[0459.0.25.25]：-/-

[0460.0.0.0] seen in disclosing of [0460.0.0.25] these paragraphs

[0461.0.25.25] embodiment 10: clone SEQ ID NO:28606 is used for expressing plant

[0462.0.0.25]: see [0462.0.0.0]

[0463.025.25] passes through pcr amplification SEQ ID NO:28606 described in Pfu Turbo or DNA Herculase polysaccharase (Stratagene) schedule of operation.

[0464.0.0.25] to[0466.0.0.25]: see that [0464.0.0.0] is to [0466.0.0.0]

[0467.025.25] selects following primer sequence for genes of SEQ ID NO:28606:

I) forward primer (SEQ ID NO:28696)

atgaaacatc?tgcatcgatt?ctttag

Ii) reverse primer (SEQ ID NO:28697)

ttaaactgat?ggacgcaaac?gaacg

[0468.0.0.25] to [0479.0.0.25]: see that [0468.0.0.0] is to [0479.0.0.0]

[0480.025.25]: embodiment 11: express the generation of the transgenic plant of SEQ ID NO:28606

[0481.0.0.25] to [0513.0.0.25]: see that [0481.0.0.0] is to [0513.0.0.0]

[0514.0.9.9]: alternatively, as Sonnebald etc., (Nat Biotechnol.1997Aug; 15 (8): 794-7) or Panikulangara etc., Plant Physiol.2004Oct; 136 (2): detect carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose described in the 3148-58.

The different plants of being analyzed the results are shown in following table 1:

Table 1

??ORF	Metabolite	Analyte	Method	??Min	??Max
						??b0019	Sucrose	Sucrose	??GC	??1.22	??1.55
??b0138	Inositol	Inositol	??GC	??1.32	??1.46
						??b0161	Fructose	Fructose	??GC	??1.82	??12.55
??b0161	Glucose	Glucose	??GC	??1.86	??4.38
						??b0161	Raffinose	Raffinose	??LC	??2.28	??2.97
??b0252	Starch/Mierocrystalline cellulose	Anhydroglucose	??GC	??1.37	??1.53
						??b0290	Inositol	Inositol	??GC	??1.23	??1.50
??b0695	Fructose	Fructose	??GC	??1.69	??11.46
						??b0730	Raffinose	Raffinose	??LC	??1.93	??7.16
??b1430	Starch/Mierocrystalline cellulose	Anhydroglucose	??GC	??1.31	??1.58
						??b1693	Starch/Mierocrystalline cellulose	Anhydroglucose	??GC	??1.34	??2.16
??b1701	Raffinose	Raffinose	??LC	??1.57	??10.58

??b1708	Fructose	Fructose	??GC	??1.77	??27.64
						??b1708	Glucose	Glucose	??GC	??1.62	??10.42
??b1886	Raffinose	Raffinose	??LC	??1.64	??4.91
						??b1926	Fructose	Fructose	??GC	??1.86	??2.93
??b1926	Glucose	Glucose	??GC	??1.63	??1.88
						??b2023	Inositol	Inositol	??GC	??1.31	??2.10
??b2597	Fructose	Fructose	??GC	??1.90	??2.05
						??b2599	Glucose	Glucose	??GC	??1.65	??2.71
??b2664	Fructose	Fructose	??GC	??1.77	??41.86
						??b2664	Raffinose	Raffinose	??LC	??1.72	??13.67
??b2699	Inositol	Inositol	??GC	??1.86	??7.90
						??b2699	Raffinose	Raffinose	??LC	??1.61	??25.08
??b3172	Inositol	Inositol	??GC	??1.26	??2.44
						??b3231	Starch/Mierocrystalline cellulose	Anhydroglucose	??GC	??1.31	??1.74
??b3430	Inositol	Inositol	??GC	??1.34	??2.16
						??b3601	Raffinose	Raffinose	??LC	??1.84	??2.35
??b4129	Inositol	Inositol	??GC	??1.29	??1.48
						??b4239	Fructose	Fructose	??GC	??4.21	??8.68

??b4239	Glucose	Glucose	??GC	??2.00	??4.85
						??b4239	Sucrose	Sucrose	??GC	??1.64	??2.69
??b4327	Fructose	Fructose	??GC	??1.97	??3.75
						??YBR184W	Raffinose	Raffinose	??LC	??1.87	??5.78
??YBR204C	Inositol	Inositol	??GC	??1.27	??2.92
						??YDR112W	Inositol	Inositol	??GC	??1.41	??1.90
??YER174C	Starch/Mierocrystalline cellulose	Anhydroglucose	??GC	??1.34	??1.84
						??YGR261C	Inositol	Inositol	??GC	??2.24	??7.98
??YGR261C	Raffinose	Raffinose	??LC	??1.95	??30.56
						??YIL150C	Inositol	Inositol	??GC	??4.80	??4.80
??YJL072C	Glucose	Glucose	??GC	??1.58	??3.93
						??YJL099W	Inositol	Inositol	??GC	??1.26	??5.72
??YOR044W	Inositol	Inositol	??GC	??1.44	??2.60
						??YOR350C	Inositol	Inositol	??GC	??1.52	??2.82
??b0050	Starch and/or Mierocrystalline cellulose	Anhydroglucose	??GC	??1.38	??1.89
						??b0124	Fructose	Fructose	??GC	??2.00	??30.26
??b0149	Fructose	Fructose	??GC	??1.68	??1.95
						??b1318	Fructose	Fructose	??GC	??2.03	??2.99

??b1463	Fructose	Fructose	??GC	??1.92	??5.53
						??b1463	Glucose	Glucose	??GC	??1.57	??7.18
??b1463	Inositol	Inositol	??GC	??1.36	??1.96
						??b1539	Starch and/or Mierocrystalline cellulose	Anhydroglucose	??GC	??1.39	??1.92
??b1736	Glucose	Glucose	??GC	??1.61	??2.10
						??b1961	Inositol	Inositol	??GC	??1.25	??1.58
??b2491	Fructose	Fructose	??GC	??2.23	??3.78
						??b2491	Glucose	Glucose	??GC	??1.81	??3.89
??b3260	Fructose	Fructose	??GC	??1.87	??5.72
						??b3578	Fructose	Fructose	??GC	??1.89	??2.06
??b3578	Glucose	Glucose	??GC	??1.65	??2.10
						??b3578	Raffinose	Raffinose	??LC	??1.55	??1.84
??b3619	Fructose	Fructose	??GC	??1.61	??2.11
						??b3619	Glucose	Glucose	??GC	??1.60	??2.19
??b3919	Starch and/or Mierocrystalline cellulose	Anhydroglucose	??GC	??1.33	??1.52
						??b4074	Inositol	Inositol	??GC	??1.25	??1.50
??b4122	Fructose	Fructose	??GC	??1.53	??6.02
						??b4232	Starch and/or Mierocrystalline cellulose	Anhydroglucose	??GC	??1.35	??1.61

?YHR072W-A

Inositol

Inositol

??GC

??1.27

??1.53

[0515.0.0.25] to [0552.0.0.25]: see that [0515.0.0.0] is to [0552.0.0.0]

[0552.1.0.1]: embodiment 15: from Zea mays metabolite profile information

As described in embodiment 14c, transform the Zea mays plant.

The different Zea mays plants of being analyzed the results are shown in following table 2:

Table 2

The ORF title	Metabolite	??Min	??Max
				??b2699	Raffinose	??2.06	??5.58
??YIL150C	Inositol	??2.03	??2.91

Table 2 shows that proline(Pro) and glutamine have increased in the genetic modification maize plant of expressing yeast saccharomyces cerevisiae nucleotide sequence YIL150c and/or Escherichia coli protein b2699.

In one embodiment, under the situation that yeast saccharomyces cerevisiae protein YIL150C or its homologue are enhanced as the activity of " S phase (DNA synthetic) initial or finish necessary chromobindins " or its homologue in maize plant, preferably, giving the fine chemicals inositol is increased between 103% and 191%.

Escherichia coli protein b2699 or have proteolytic enzyme and the DNA chain exchange of nuclease and recombinant protein, preferably have under the situation that the activity of the active gene product of recombinant protein recA superfamily or its homologue is enhanced in maize plant, preferably, giving the fine chemicals raffinose is increased between 106% and 458%.

[0552.2.4.4]: see [0552.2.0.0]

[0553.0.25.25]

1. produce carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably method of sucrose, it comprises:

(a) in non-human being or its one or more parts, improve or produce as Table II the 5th or 7 is listed as, is respectively capable for starch and/or cellulosic 290-294 and/or 604-607; 295-302 and/or 608-616 for fructose are capable; 303-308 and/or 617-621 for glucose are capable; 309-322 and/or 622-625 for inositol are capable; 323-331 and/or 626 row for raffinose; For the 332-333 of sucrose capable shown in the activity of protein or its functional equivalents; With

(b) in allowing described biology, produce respectively carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably cultivate this biology under the condition of sucrose.

2. produce carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably method of sucrose, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding as Table II the 5th or 7 be listed as, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively; 295-302 and/or 608-616 for fructose are capable; 303-308 and/or 617-621 for glucose are capable; 309-322 and/or 622-625 for inositol are capable; 323-331 and/or 626 row for raffinose; For the 332-333 of sucrose capable shown in polypeptide or its fragment, described nucleic acid molecule give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases;

B) contain just like Table I the 5th or 7 row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively; 295-302 and/or 608-616 for fructose are capable; 303-308 and/or 617-621 for glucose are capable; 309-322 and/or 622-625 for inositol are capable; 323-331 and/or 626 row for raffinose; For the 332-333 of sucrose capable shown in the nucleic acid molecule of nucleic acid molecule;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity;

E) under stringent hybridization condition, hybridize and give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprise by use as Table III the 7th is listed as, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively; 295-302 and/or 608-616 for fructose are capable; 303-308 and/or 617-621 for glucose are capable; 309-322 and/or 622-625 for inositol are capable; 323-331 and/or 626 row for raffinose; For the 332-333 of sucrose capable shown in primer or primer nucleic acid molecule that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that vin quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, capable for starch and/or cellulosic 290-294 and/or 604-607 respectively; 295-302 and/or 608-616 for fructose are capable; 303-308 and/or 617-621 for glucose are capable; 309-322 and/or 622-625 for inositol are capable; 323-331 and/or 626 row for raffinose; For the 332-333 of sucrose capable shown in consensus sequence polypeptide and give biology or its part in carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give each fine chemicals quantity increase in biology or its part, wherein said probe fragment has at least 15 nt, preferred 20 nt, 30nt, 50 nt, 100 nt, 200 nt or 500 nt of (a) to (k) institute characterisation of nucleic acids molecule;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, wherein carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose is separated.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) reclaim and free that optionally separating is produced by selected mutation biology or its part or bonded carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding as Table II the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in polypeptide or its fragment, described nucleic acid molecule give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases;

B) contain just like Table I the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in the nucleic acid molecule of nucleic acid molecule;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity;

E) under stringent hybridization condition, hybridize and give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose quantity with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by using as Table III the 7th row, it is capable to be respectively 290-294 and/or 604-607,295-302 and/or 608-616 are capable, 303-308 and/or 617-621 are capable, 309-322 and/or 622-625 are capable, 323-331 and/or 626 row, the nucleic acid molecule that primer or primer shown in 332-333 is capable obtain amplifier nucleic acid molecule from cDNA library or genomic library, and give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases;

H) nucleic acid molecule, its coding contain just like Table IV the 7th row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 row, 332-333 capable shown in consensus sequence polypeptide and give biology or its part in carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases, and wherein said probe fragment has at least 15 nt of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt;

Thus, nucleic acid molecule by one or more Nucleotide be different from as Table I A the 5th or 7 row, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 the row, 332-333 capable shown in sequence.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, its method by as claim 13 described in produces or by the nucleic acid molecule encoding as claim 6 described in, thus this polypeptide by one or more amino acid be different from as Table II A the 5th or 7 be listed as, be respectively 290-294 and/or 604-607 is capable, 295-302 and/or 608-616 is capable, 303-308 and/or 617-621 is capable, 309-322 and/or 622-625 is capable, 323-331 and/or 626 capable, 332-333 capable shown in sequence.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the agonist of screening polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose quantity increases, described method comprises:

(a) will express by the nucleic acid molecule of claim 6 coded and give carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably cell, tissue, plant or microorganism and the candidate compound of the polypeptide that increases of sucrose quantity or the sample that comprises multiple compound contact under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps cultivate or keep carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level or expression of polypeptides level in the substratum of cell, tissue, plant or microorganism; With

(c) carbohydrate by measuring, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level or expression of polypeptides level and the standard carbohydrate of when described candidate compound or the sample that comprises described multiple compound lack, measuring, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose or expression of polypeptides level are relatively identified agonist or antagonist; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify give carbohydrate in plant or the microorganism, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose produces the method for the compound that improves, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably the polypeptide that increases of sucrose quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises described read-out system and polypeptide interaction in the presence of the sample of multiple compound, and it is coded and give carbohydrate in biology or its part to be provided at the nucleic acid molecule of described read-out system of permission and claim 6, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably respond compound and described polypeptide bonded detectable signal under the condition of the expression of polypeptides of sucrose quantity increase; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify give carbohydrate in the cell, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose produces the method for the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain to be coded in gives carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably candidate gene of the gene product that increases of sucrose after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule import to be fit to is produced carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably host cell of sucrose;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) analyze carbohydrate in the host cell, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level; With

(f) identify nucleic acid molecule and gene product thereof, compare with wild-type, give after it is expressed carbohydrate in the host cell, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level improves.

20. identify give carbohydrate in the cell, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose produces the method for the gene product that improves, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain to be coded in gives carbohydrate in biology or its part, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably candidate gene of the gene product that improves of sucrose quantity or level after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule import to be fit to is produced carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably host cell of sucrose;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) analyze carbohydrate in the host living beings, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level; With

(e) identify nucleic acid molecule and gene product thereof, compare with wild-type, give after it is expressed carbohydrate in the host cell, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose level improves.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably nucleic acid molecule that increases of sucrose after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying and can gives regulation and control carbohydrate, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, the more preferably compound of sucrose level at biology.

25. makeup, medicine, food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the nucleic acid molecule of claim 6, the polypeptide of claim 14, the nucleic acid construct of claim 7, claim 8 or 9 carrier, antagonist or agonist according to claim 17 evaluation, the antibody of claim 15, the plant of claim 16 or plant tissue, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make the anti-carbohydrate that suppresses of plant, preferred polysaccharide, more preferably starch and/or Mierocrystalline cellulose and/or monose, more preferably fructose, glucose and/or inositol and/or trisaccharide, more preferably raffinose and/or disaccharides, more preferably sucrose synthetic weedicide.

[0554.0.0.25] summary: see [0554.0.0.0]

Claims (30)

1. produce the method for each fine chemicals, it comprises:

(a) in the activity that improves or produce shown in Table II the 5th or 7 row protein or its function equivalent in non-human being or its one or more parts; With

(b) in allowing described biology, cultivate this biology under the condition of each fine chemicals of generation.

2. produce the method for each fine chemicals, be included in to improve in biology or its part or produce and express at least a nucleic acid molecule that is selected from following nucleic acid molecule that contains:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, and described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives that each fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that each fine chemicals quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row and give biology or its part in each fine chemicals quantity increase; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give each fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Or contain the nucleic acid molecule of above-mentioned nucleic acid molecule complementation sequence.

3. claim 1 or 2 method, it comprises and reclaims free or each fine chemicals of bonded.

4. each described method in the claim 1 to 3, it may further comprise the steps:

(a) select expression biological or its part by the polypeptide of claim 2 a characterisation of nucleic acids molecule encoding;

(b) with selected biology or its part mutagenesis;

(c) activity or the expression level of polypeptide described in the activity of polypeptide or expression level and selected biology or its part described in biological or its part compare with mutagenesis;

(d) select to compare the described polypeptide active that contains raising or institute's mutation biology or its part of expression level with selected biology or its part;

(e) randomly, cultivate and cultivate this biology or its part; With

(f) recovery and optionally separating are by free or each fine chemicals of bonded of selected mutation biology or the generation of its part.

5. each described method in the claim 1 to 4, the wherein instantaneous or expression that stably improves or produce described activity of proteins or described nucleic acid molecule.

6. contain the isolated nucleic acid molecule that is selected from following nucleic acid molecule:

A) nucleic acid molecule, coding is polypeptide or its fragment shown in Table II the 5th or 7 row, and described nucleic acid molecule is given each fine chemicals quantity increase in biology or its part;

B) contain just like the nucleic acid molecule of nucleic acid molecule shown in Table I the 5th or 7 row;

C) since the degeneracy of genetic code can from nucleic acid molecule (a) or (b) coded peptide sequence derive its sequence and give biology or its part the nucleic acid molecule that increases of each fine chemicals quantity;

D) the coded amino acid sequence of polypeptide of coded polypeptide and nucleic acid molecule (a) to (c) have at least 50% identity and give biology or its part in the nucleic acid molecule that increases of each fine chemicals quantity;

E) under stringent hybridization condition, hybridize and give the nucleic acid molecule that each fine chemicals quantity increases in biology or its part with nucleic acid molecule (a) to (c);

F) nucleic acid molecule, it comprises by the nucleic acid molecule that uses shown in Table III the 7th row primer or primer that amplifier nucleic acid molecule from cDNA library or genomic library is obtained, and gives that each fine chemicals quantity increases in biology or its part;

G) nucleic acid molecule, its coding by means of at nucleic acid molecule (a) to the monoclonal antibody of one of (f) coded polypeptide and isolated polypeptide, and give that each fine chemicals quantity increases in biology or its part;

H) nucleic acid molecule, its coding contain just like the polypeptide of consensus sequence shown in Table IV the 7th row and give each fine chemicals quantity in biological or its part to be increased; With

I) nucleic acid molecule, it can obtain by probe or the suitable nucleic acid library of its fragment screening with one of sequence that contains nucleic acid molecule (a) to (k) under stringent hybridization condition, and give each fine chemicals quantity increase in biology or its part, wherein said probe fragment has the 15nt at least of (a) to (k) institute characterisation of nucleic acids molecule, preferred 20nt, 30nt, 50nt, 100nt, 200nt or 500nt;

Thus, nucleic acid molecule is different from sequence shown in Table I A the 5th or 7 row by one or more Nucleotide.

7. nucleic acid construct, it contains one or more regulatory elements, and entitle requires 6 nucleic acid molecule to express.

8. carrier, it contains the nucleic acid construct just like nucleic acid molecule described in the claim 6 or claim 7.

9. the carrier described in claim 8, wherein nucleic acid molecule with regulate sequence and effectively be connected so that in protokaryon or eucaryon host or protokaryon and eucaryon host, express.

10. host cell, its stable or instantaneous conversion carrier described in claim 8 or 9 or the nucleic acid construct of nucleic acid molecule described in claim 6 or claim 7, perhaps as in the claim 2 to 5 as described in each and produce.

11. the host cell of claim 10, it is a genetically modified host cell.

12. the host cell of claim 10 or 11, it is vegetable cell, zooblast, microorganism or yeast cell, fungal cell, prokaryotic cell prokaryocyte, eukaryotic cell or archeobacteria.

13. produce the method for polypeptide, wherein polypeptide is as expressing in each described host cell in the claim 10 to 12.

14. polypeptide, it produces by the method described in claim 13 or by the nucleic acid molecule encoding described in claim 6, this polypeptide is different from sequence shown in Table II A the 5th or 7 row by one or more amino acid thus.

15. antibody, it combines with polypeptid specificity described in claim 14.

16. plant tissue, reproductive material, results material or plant, it contains just like the host cell described in the claim 12, and described host cell is vegetable cell or Agrobacterium.

17. the screening agonist of polypeptide active and the method for antagonist, described polypeptide by the nucleic acid molecule encoding of claim 6 and give biology or its part in each fine chemicals quantity increase, described method comprises:

(a) sample that will express and cell, tissue, plant or the microorganism of giving in biology or its part polypeptide that each fine chemicals quantity increase coded by the nucleic acid molecule of claim 5 and candidate compound or comprise multiple compound contacts under the condition of this expression of polypeptides of permission;

(b) measure in cell, tissue, plant or the microorganism, perhaps each fine chemicals level or the expression of polypeptides level in the substratum of cultivation or maintenance cell, tissue, plant or microorganism; With

(c) relatively identify agonist or antagonist by each fine chemicals level that will measure or expression of polypeptides level and each fine chemicals of standard or the expression of polypeptides level when described candidate compound or the sample that comprises described multiple compound lack, measured; The level that improves than the standard sample that this compound is described or comprises described multiple compound is an agonist thus, and the sample that this compound is described or comprises described multiple compound than the level that standard reduces is an antagonist.

18. identify the method for giving the compound that each fine chemicals generation improves in plant or the microorganism, said method comprising the steps of:

(a) culturing plants cell or tissue or microorganism or keep plant under proper condition, these vegetable cells or tissue or microorganism or expression of plants by the nucleic acid molecule encoding of claim 6 and give biology or its part in the polypeptide that increases of each fine chemicals quantity and express can with the interactional read-out system of polypeptide, described felicity condition allows at compound or comprises that described read-out system interacts with polypeptide in the presence of the sample of multiple compound, and it is coded and give under the condition of the expression of polypeptides of each fine chemicals quantity increase in biology or its part and respond compound and described polypeptide bonded detectable signal to be provided at the nucleic acid molecule that allows described read-out system and claim 6; With

(b) existence by detecting the signal that described read-out system produces or do not exist or improve and come whether authenticating compound is effective agonist.

19. identify the method for giving the gene product that each fine chemicals generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule with claim 6 contacts with sample nucleic acid molecule, and described sample nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that each fine chemicals increases after the expression;

(b) identify under lax stringent condition nucleic acid molecule with the making nucleic acid molecular hybridization of claim 6;

(c) the candidate nucleic acid molecule is imported the host cell that is fit to produce each fine chemicals;

(d) in host cell, express the nucleic acid molecule of being identified;

(e) each fine chemicals level in the analysis host cell; With

(f) identify nucleic acid molecule and gene product thereof, compare, give each fine chemicals level raising in the host cell after it is expressed with wild-type.

20. identify the method for giving the gene product that each fine chemicals generation improves in the cell, said method comprising the steps of:

(a) nucleic acid molecule of identification of organism in database, described nucleic acid molecule can contain and is coded in the candidate gene of giving the gene product that each fine chemicals quantity in biology or its part or level improve after the expression, and with nucleic acid molecule at least 20% homology of claim 6;

(b) the candidate nucleic acid molecule is imported the host cell that is fit to produce each fine chemicals;

(c) in host cell, express the nucleic acid molecule of being identified;

(d) each fine chemicals level in the analysis host living beings; With

(e) identify nucleic acid molecule and gene product thereof, compare, give each fine chemicals level raising in the host cell after it is expressed with wild-type.

21. produce the method for Pestcidal compositions, described method comprises the step of each described method in the claim 17 to 20, but and each compounds identified in the claim 17 to 20 is mixed with the form of agricultural application.

22. composition, antagonist that it contains in the nucleic acid construct, claim 8 or 9 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 each carrier, identify according to claim 17 or agonist, compound, claim 19 or 20 the gene product of claim 18, the antibody of claim 15, and randomly contain can the agricultural carrier.

23. the purposes of the nucleic acid molecule described in claim 6, it is used to identify gives the nucleic acid molecule that each fine chemicals increases after expression.

24. the polypeptide of claim 14 or the nucleic acid construct of claim 7 or according to the purposes of the method genes identified product of claim 19 or 20, it is used for identifying can give the compound of each fine chemicals level of regulation and control at biology.

25. food or feed composition, the results material of the antibody of antagonist that it contains the nucleic acid construct, claim 8 of polypeptide, the claim 7 of nucleic acid molecule, the claim 14 of claim 6 or 9 carrier, identify according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the method genes identified product of claim 19 or 20.

26. the results material of the antibody of the carrier of the nucleic acid construct of the nucleic acid molecule of claim 6, the polypeptide of claim 14, claim 7, claim 8 or 9, the antagonist of identifying according to claim 17 or agonist, claim 15, the plant of claim 16 or plant tissue, claim 16, the host cell of claim 10 to 12 or according to the purposes of the method genes identified product of claim 19 or 20, it is used to make anti-each fine chemicals synthetic weedicide that suppresses of plant.

27. vegetable cell, the vegetable cell device, plant tissue, plant or its part, wherein with before 2 days June in 2005, be preserved in (the Institut f ü rPflanzengenetik und Kulturpflanzenforschung (IPK) of German plant genetics and cultivated plant institute, Corrensstra β e 3, D-06466 Gatersleben, Germany) each fine chemicals content is compared in mutation increases by 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, 1000%, 5000%, 10000%, 50000%, 100000% or more, and the content meaning is meant with respect to vegetable cell, the vegetable cell device, plant tissue, the weight of each fine chemicals of dry weight of plant or its part, and the dry weight meaning is meant vegetable cell, the vegetable cell device, plant tissue, plant or its part deduct vegetable cell, the vegetable cell device, plant tissue, the weight of the organic substance in plant or its part after institute's water content.

28. the vegetable cell of claim 27, vegetable cell device, plant tissue, plant or its part, wherein the increase of each fine chemicals content is by improving or producing shown in Table II A and B the 5th or 7 row protein or its functional equivalent or comprise as shown in Table IV that the activity of proteins of consensus sequence realizes.

29. vegetable cell, vegetable cell device, plant tissue, plant or its part, wherein be selected from following mutation and compare each fine chemicals content and increase by 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200%, 300%, 500%, 1000%, 5000%, 10000%, 50000%, 100000% or more:

(a) upland cotton (G.hirsutum) IPK accession number GOS 6 (D 120), GOS 7 (ST 446), GOS 10 (D 1635), GOS 17 (D 4302) or GOS 21 (D 5553), perhaps Somalia cotton (G.areysianum Deflers), perhaps G.incanum (Schwartz) Hillc., perhaps Lei Mengdeshi cotton (G.raimondii Ulbr.), perhaps Arabic cotton (G.stocksii Masters), perhaps plucked instrument Bai Shi cotton (G.thurberi Tod.), perhaps Hawaii cotton (G.tomentosum Nutt.), perhaps three leaf cottons (G.triphyllum Hochr.), perhaps tree cotton (Gossypium arboretum) IPK accession number GOS 13 (D 1634), GOS 16 (D 4240), GOS 18 (D 4505), GOS 19 (D4506), GOS 20 (D 4750) or GOS 12 (D 1329), perhaps sea island cotton (Gossypiumbarbadense), perhaps cotton (Gossypium herbaceum); With

(b) colea (Brassica napus) Mika mutation, colea Digger mutation, colea Artus mutation, colea Terra mutation, colea Smart mutation, colea Olivine mutation, colea Libretto mutation, colea Wotan mutation, colea Panther mutation, colea Express mutation, colea Oase mutation, colea Elan mutation, colea Ability mutation, colea Mohican mutation; With

(c) flax (Linum usitatissimum) Librina mutation, flax Flanders mutation, flax Scorpion mutation, flax Livia mutation, flax Lola mutation, flax Taurus mutation, flax Golda mutation, flax Lirima mutation; With

(d) Zea mays (Zea mays) Articat mutation, Zea mays NK Dilitop mutation, Zea mays Total mutation, Zea mays Oldham mutation, Zea mays Adenzo mutation, Zea mays NKLugan mutation, Zea mays Liberal mutation, Zea mays Peso mutation; With

(e) soybean (Glycine max) Oligata mutation, soybean Lotus mutation, soybean Primus mutation, soybean Alma Ata mutation, soybean OAC Vision mutation, soybean Jutro mutation; With

(f) Sunflower Receptacle (Helianthus annus) Helena mutation, Sunflower Receptacle Flavia mutation, Sunflower Receptacle Rigasol mutation, Sunflower Receptacle Flores mutation, Sunflower Receptacle Jazzy mutation, Sunflower Receptacle Pegaso mutation, Sunflower Receptacle Heliaroc mutation, Sunflower Receptacle Salut RM mutation; With

(g) flax shepherd's purse (Camelina sativa) Dolly mutation, flax shepherd's purse Sonny mutation, flax shepherd's purse Ligena mutation, flax shepherd's purse Calinka mutation; With

(h) sinapsis alba (Sinapis alba) Martigena mutation, sinapsis alba Silenda mutation, sinapsis alba Sirola mutation, sinapsis alba Sito mutation, sinapsis alba Semper mutation, sinapsis alba Seco mutation; With

(i) safflower (Carthamus tinctorius) Sabina mutation, safflower HUS-305 mutation, safflower landrace mutation, safflower Thori-78 mutation, safflower CR-34 mutation, safflower CR-81 mutation; With

(j) mustard Lay (Brassica juncea) Vittasso mutation, mustard Lay Muscon M-973 mutation, mustard Lay RAPD mutation, mustard Lay Co.J.86 mutation, mustard Lay IAC 1-2 mutation, mustard Lay Pacific Gold mutation; With

(k) coconut (Cocos nucifera L.) Maypan, Ceylon Tall, Indian Tall, JamaicaTall, Malayan Tall, Java Tall, Laguna, King CRIC 60, CRIC 65, CRISL 98, Moorock tall, Plus palm tall, San Ramon, Typica, Nana or Aurantiaca mutation; With

(l) common wheat (Triticum aestivum L.) Altos mutation, Bundessortenamt submits numbering 2646, common wheat Bussard mutation to, Bundessortenamt submits numbering 1641 or common wheat Centrum mutation to, and Bundessortenamt submits numbering 2710 to; With

(m) beet (Beta vulgaris) Dieck 13 mutation, CPVO submits numbering 19991828, beet FD 007 mutation to, and CPCO submits numbering 20000506 or beet HI 0169 mutation to, and CPVO submits numbering 20010315 to; With

(n) barley (Hordeum vulgare) Dorothea mutation, CPVO submits numbering 20031457, barley Colibri mutation to, CPVO submits numbering 20040122, barley Brazil mutation to, and CPVO submits numbering 20010274 or barley Christina mutation to, and CPVO submits numbering 20030277 to; With

(o) rye (Secale cereale) Esprit mutation, CPVO submits numbering 19950246, rye Resonanz mutation to, and CPVO submits numbering 20040651 or rye Ursus mutation to, and CPVO submits numbering 19970714 to; With

(p) rice (Oryza sativa) Gemini mutation, CPVO submits numbering 20010284, rice Tanaro mutation to, and CPVO submits numbering 20020177 or rice Zeus mutation to, and CPVO submits numbering 19980388 to; With

(q) Ma Lingzhu (S0lanum tuberosum L.) Linda, Nicola, Solara, Agria, Sieglinde or Russet Burbank mutation; With

(r) Semen arachidis hypogaeae (Arachis hypogaea) fastigiata subspecies Valencia cultivated variety; With

(s) Semen arachidis hypogaeae hypogaea subspecies Virginia cultivated variety " Holland Jumbo ", " Virginia A23-7 " or " Florida 416 " mutation; With

(t) Semen arachidis hypogaeae hirsuta subspecies Peruvian runner cultivated variety " Southeastern Runner56-15 ", " Dixie Runner " or " Early Runner " mutation; With

(u) Semen arachidis hypogaeae vulgaris subspecies Spanish cultivated variety " Dixie Spanish ", " Improved Spanish 2B " or " GFA Spanish " mutation;

And the content meaning is meant the weight of each fine chemicals for the dry weight of vegetable cell, vegetable cell device, plant tissue, plant or its part, and the dry weight meaning is meant that vegetable cell, vegetable cell device, plant tissue, plant or its part deduct the weight of the organic substance after institute's water content in vegetable cell, vegetable cell device, plant tissue, plant or its part.

30. the vegetable cell of claim 29, vegetable cell device, plant tissue, plant or its part, wherein the increase of each fine chemicals content is by improving or producing shown in Table II A and B the 5th or 7 row protein or its functional equivalent or comprise as shown in Table IV that the activity of proteins of consensus sequence realizes.

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