CN101054593A

CN101054593A - Corynebacterium glutamicum gene encoding hpr of phosphoenolpyruvate:sugar phosphotransferase system

Info

Publication number: CN101054593A
Application number: CNA2007100920742A
Authority: CN
Inventors: M·波姆佩朱斯; B·克雷格尔; H·施雷德尔; O·策尔德; G·哈贝豪尔
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 1999-07-01
Filing date: 2000-06-27
Publication date: 2007-10-17
Also published as: ZA200200816B; CN101054592A; CN101130778A

Abstract

Isolated nucleic acid molecules, designated PTS nucleic acid molecules, which encode novel PTS proteins from Corynebacterium glutamicum are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PTS nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated PTS proteins, mutated PTS proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C. glutamicum based on genetic engineering of PTS genes in this organism.

Description

Orynebacterium: the Corynebacterium glutamicum gene of sugar phosphotransferase system proteins

The application is the divisional application for the Chinese patent application 00812165.6 " Orynebacterium: the Corynebacterium glutamicum gene of sugar phosphotransferase system proteins " that the applying date is on June 27th, 2000.

Technical field

The present invention relates to the nucleic acid molecules of the new Corynebacterium glutamicum pts protein of the coding of separation.The present invention also relates to antisense nucleic acid molecule, the recombinant expression carrier containing PTS nucleic acid molecules, and imported the host cell of expression vector.The present invention is also further to isolated pts protein, PTS mutain, fused protein, Antigenic Peptide, and the method for improving the required production of chemicals carried out by the organism based on Corynebacterium glutamicum PTS gene genetic engineering.

Background technique

Specific product and by-product in cell in naturally occurring metabolic process have purposes, including food, feed, cosmetics and pharmaceutical industries in many industries.These molecules are collectively referred to as " fine chemical ", including organic acid, protein sources and amino acid, nucleotide and the nucleosides in non-protein source, lipid and fatty acid, glycol, carbohydrate, aromatic compound, vitamin and co-factor and enzyme.The bacterium of a large amount of specific required molecules can be generated and secreted by large-scale culture, and most convenient prepares these products.A kind of particularly useful organism for this purpose is exactly Corynebacterium glutamicum (Corynebacterium glutamicum), a kind of Gram-positive nonpathogenic bacteria.By bacterial strain screening, there are many mutant strains for generating large quantities of required compounds.However, the bacterial strain screening carried out to improve particular molecule production, is the process an of time-consuming and difficulty.

Summary of the invention

The present invention provides new bacterial nucleic acid molecule, these molecules serve many purposes.These purposes include that identification can produce the microorganism of fine chemical, the generation for adjusting Corynebacterium glutamicum or the fine chemical in relationship bacterium, the parting of Corynebacterium glutamicum or relationship bacterium and identification, as the reference point for drawing Corynebacterium glutamicum gene group map.These new nucleic acid molecule encoding protein, referred to herein as phosphoenolpyruvate: sugar phosphotransferase system (PTS) protein.

Corynebacterium glutamicum is a kind of Gram-positive aerobic bacterium, usually be used to that various fine chemicals be mass produced in the industry, is also used for degradation of hydrocarbon (such as in Oil spills) and oxidation terpineol.Therefore, PTS nucleic acid molecules of the invention can be used to identify the microorganism that can be used to produce fine chemical, such as pass through fermentation process.Adjust the expression of PTS nucleic acid molecules of the present invention, or the sequence of modification PTS nucleic acid molecules of the present invention, it can be used for adjusting the production (for example, the generation for improving one or more kinds of fine chemicals in bar bacterium or brevibacterium) of one or more kinds of fine chemicals in microorganism.

PTS nucleic acid molecules of the present invention can be used for identifying a kind of microorganism whether be Corynebacterium glutamicum perhaps its relationship bacterial strain or identification microorganism population mixture Glutamic Acid bar bacterium or its relationship bacterial strain presence.The present invention provides the nucleic acid sequences of many Corynebacterium glutamicum genes；Under strict conditions, the genomic DNA extracted from single microorganism or mixed microorganism culturing in groups object is detected with probe, which covers the distinctive one section of region of Corynebacterium glutamicum gene, may determine whether with the presence of the microorganism.Although Corynebacterium glutamicum itself be it is non-pathogenic, it is related with the pathogen species in human body, such as Corynebacterium diphtheriae bacterium (Corynebacterium diphtheriae) (diphtheria cause a disease original)；This microorganism is detected with great Clinical practicability.

PTS nucleic acid molecules of the present invention are also used as drawing the reference point of Corynebacterium glutamicum gene group map, or draw the reference point of its relationship strain gene group map.Similar, perhaps its variant or part thereof may be used as the genetic marker of genetic engineering bar bacterium or brevibacterium to these molecules.

It, can be seeming that high-energy carbon-containing molecules as glucose are transported into Corynebacterium glutamicum, or participate in the Cellular Signaling Transduction Mediated in the microorganism for example, the pts protein of novel nucleic acids molecule encoding of the present invention.In view of the practicability for the cloning vector that can be used in corynebacterium glutamicum, such as in Sinskey et al., US patent number No.4,649, disclosed in 119, and consider Genetic Manipulative Technology (Yashihama the et al, J.Bacteriol.162:591-597 (1985) of Corynebacterium glutamicum and relationship brevibacterium strain (such as brevibacterium)；Katsumata et al., J.Bacteriol.159:306-311 (1984)；And Santamaria et al., J.Gen.Microbiol.130:2237-2246 (1984)), nucleic acid molecules of the invention can be used for the genetic engineering of the organism, make that one or more kinds of fine chemicals are better or the more effective producer.

PTS molecule of the invention can be modified, so that yield, production and/or the production efficiency of one or more kinds of fine chemicals are improved.For example, by modifying a kind of pts protein for participating in glucose uptake its activity can be optimized, so that the rate that glucose uptake quantity or glucose are transported into cell is improved.Glucose inside cells or other carbohydrate degradations can provide energy, the biochemical reaction that these energy can be used for pushing energy unfavorable, such as those are related to the reaction of fine chemical biosynthesis.Degradation also provides intermediate or precursor molecule necessary to certain fine chemical biosynthesis, such as amino acid, vitamin and co-factor.By modifying PTS molecule of the present invention to increase the quantity of intracellular high energy carbon molecules, not only it can increase the energy supply of metabolic pathway necessary to completing to produce one or more kinds of fine chemicals, but also endocellular metabolism substance library required for this production can be increased.

In addition, PTS molecule of the present invention can participate in one or more of Cellular Signaling Transduction Mediated approach, these signal transduction paths can influence the yield and/or throughput rate of one or more kinds of fine chemicals in Corynebacterium glutamicum.Such as, once there is sufficient amount of carbohydrate into the cell, from inputted in extracellular medium protein necessary to one or more kinds of carbohydrates (such as, HPr, Enzyme I, or one of Enzyme II complex ingredient) be often translated after modify, to prevent them from sugar is input into the cell again.The quantity of sugar when movement system is closed, for maintain may be for cell normal function it is enough, this may limit the excessive production of required chemical substance.Therefore, it modifies pts protein of the present invention and it is made to be very worthwhile there is no reaction to this negative regulator, so, intracellular one or more kinds of sugar are allowed to reach higher concentration, and extended through reaction, it, can significantly more efficient production or the one or more kinds of fine chemicals of more large-tonnage acquisition from the organism containing this pts protein mutant.

The present invention provides the nucleic acid molecules of new coding protein, this protein is referred to herein as phosphoenolpyruvate: sugar phosphotransferase system (PTS) protein, they participate in high energy carbon molecules (such as, glucose, fructose, sucrose) input Corynebacterium glutamicum, and/or participate in one or more of Corynebacterium glutamicum Cellular Signaling Transduction Mediated approach.The example of these protein, the protein including those coded by said gene listed in table 1.

Therefore, one aspect of the present invention is related to, separate containing a kind of one section of nucleic acid molecules for encoding pts protein or the nucleic acid sequence of its biologically-active moiety (such as, cDNA, DNA, perhaps RNA) and separation be suitable as detect or expand PTS code nucleic acid (such as DNA or RNA) primer or hybridization probe nucleic acid fragment.In particularly preferred embodiments, isolated nucleic acid molecules include one section of Serial No. odd number being listed in sequence table nucleic acid sequence (such as, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) an either this nucleotide sequence coding region or its complementary series.In other particularly preferred embodiments, the nucleic acid molecules of the present invention of separation include with the nucleotide sequence of the Serial No. odd number in sequence table (such as, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) or its part have at least about 50% homology, preferably there is at least about 60% homology, more preferably there is at least about 70%, 80%, or 90% homology, even more preferably have at least about 95%, 96%, 97%, 98%, 99% or higher homology.In other preferred embodiments, separated nucleic acid molecule encoding is listed in the even order amino acid sequence (for example, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8) in sequence table.Currently preferred pts protein is it is also preferred that have at least one PTS activity described herein.

In another embodiment, a kind of protein of separated nucleic acid molecule encoding or its part, protein therein or its part include one section of amino acid sequence, the sequence and amino acid sequence of the invention (such as, the sequence of even order number in sequence table) there is sufficient homology, for example, having sufficient homology with amino acid sequence of the invention and making the protein or its part that there is PTS activity.It is preferred that, the protein of nucleic acid molecule encoding or its part, with the ability participated in high energy carbon molecules (for example, glucose, fructose, sucrose) input Corynebacterium glutamicum, and/or participate in the ability of one or more of Corynebacterium glutamicum Cellular Signaling Transduction Mediated approach.In one embodiment, a kind of protein of nucleic acid molecule encoding and amino acid sequence (for example, the complete amino acid sequence selected from the even order sequence in sequence table) of the invention have at least about 50% homology, preferably there is at least about 60% homology, more preferably have at least about 70%, 80%, 90% homology most preferably has at least about 95%, 96%, 97%, 98%, 99% or higher homology.In another preferred embodiment, protein is the Corynebacterium glutamicum protein of overall length, the protein and full length amino acid sequence of the invention (by be shown in corresponding sequence table odd serial numbers nucleic acid sequence (such as, SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7) open reading frame coding) sufficiently homologous.

In another preferred embodiment, isolated nucleic acid molecules come from Corynebacterium glutamicum, and encode a kind of a kind of protein (such as PTS fusion protein), the protein includes one section of bioactivity region, the region and a kind of amino acid sequence of the invention (such as, a sequence in sequence table even order sequence) there are at least about 50% or higher homology, and the protein can participate in a high energy carbon molecules (such as, glucose, fructose, sucrose) input Corynebacterium glutamicum, and/or participate in one or more of Corynebacterium glutamicum Cellular Signaling Transduction Mediated approach, or possess one or more kinds of activity being listed in Table 1, and the protein also includes the heterologous nucleic acid sequence of one section of encoding heterologous polypeptide or adjustment region.

In another embodiment, the length of at least 15 nucleotide of isolated nucleic acid molecules, and under strict conditions with contain nucleotide sequence of the present invention (for example, the odd serial numbers sequence in sequence table) making nucleic acid molecular hybridization.It is preferred that isolated nucleic acid molecules are consistent with naturally occurring nucleic acid molecules.More preferably, the naturally occurring Corynebacterium glutamicum pts protein of isolated nucleic acid molecule encoding or its biologically-active moiety.

Another aspect of the present invention is related to carrier, such as the recombinant expression carrier containing nucleic acid molecules of the present invention, and be introduced into the host cell of this carrier.In one embodiment, by being cultivated in suitable culture medium, this host cell be used to produce pts protein.Then the pts protein can be separated from culture medium or host cell.

In addition, another aspect of the present invention is related to a kind of microorganism by being genetically changed, PTS gene has been incorporated into wherein or has changed.In one embodiment, by introducing as the encoding wild type of transgenosis or the nucleic acid molecules of the present invention of saltant type PTS sequence, the genome of the microorganism is changed.In another embodiment, the endogenous PTS gene in the microbial genome is changed, for example, carrying out homologous recombination by using the PTS gene having changed and carrying out functional destruction.In another embodiment, endogenous or introducing PTS gene is changed by one or more point mutation, missing or inversion in the microorganism, but still can encoding function pts protein.In another embodiment, one or more adjustment region (for example, promoter, repressor or inducer) for changing microorganism PTS gene, to adjust the expression of PTS gene.In preferred embodiments, microorganism belongs to rod strain or quarter butt strain, particularly preferably Corynebacterium glutamicum.In preferred embodiments, also using compound needed for micro-organisms, such as amino acid, particularly preferably lysine.

On the other hand, the present invention provides corynebacterium diphtheriae presence or active methods in a kind of identification subject.This method include to one or more kinds of nucleic acid or amino acid sequence of the invention in subject (such as, be listed in the sequence of SEQ ID NO 1 to 34 in sequence table) detection, so as to detect the presence or activity of subject's Glutamic Acid bar bacterium.

In addition, another aspect of the present invention is related to separated pts protein or its part, such as its biologically-active moiety out.In a preferred embodiment, isolated pts protein or its part can participate in a high energy carbon molecules (such as, glucose, fructose, sucrose) input Corynebacterium glutamicum, and one or more of Corynebacterium glutamicum Cellular Signaling Transduction Mediated approach can also be participated in.In another preferred embodiment, separated pts protein or its part and a kind of amino acid sequence of the invention (such as, sequence table even order number a: sequence in sequence) there is sufficiently high homology, so that the protein or its part may participate in a high energy carbon molecules (such as, glucose, fructose, sucrose) ability of Corynebacterium glutamicum is inputted, and/or also assist in the ability of one or more of Corynebacterium glutamicum Cellular Signaling Transduction Mediated approach.

Present invention provides the Separation Products of pts protein.In preferred embodiments, pts protein includes amino acid sequence of the invention (for example, sequence table even order number: a sequence in sequence).In another preferred embodiment, the present invention is related with isolated full length protein, and the protein and all aminoacid sequence (sequence table even order number a: sequence in sequence) of the invention (being encoded by the open reading frame for the Serial No. odd number being shown in corresponding sequence table) have quite high homology.In addition, in another embodiment, protein and all aminoacid sequence of the invention (such as, even order sequence in sequence table) there is at least about 50% homology, preferably there is at least about 60% homology, more preferably there is at least about 70%, 80%, or 90% homology, most preferably have at least about 95%, 96%, 97%, 98% or 99% or higher homology.In another embodiment, amino acid sequence that isolated pts protein includes and an amino acid sequence of the invention (such as, an even order sequence in sequence table) there are at least about 50% or higher homology, and can participate in a high energy carbon molecules (such as, glucose, fructose, sucrose) input Corynebacterium glutamicum, with/and one or more of Corynebacterium glutamicum Cellular Signaling Transduction Mediated approach are participated in, or with the one or more activity listed in table 1.

In addition, isolated pts protein can contain the amino acid sequence by nucleic acid sequence encoding, one nucleic acid array hybridizing of the nucleic acid sequence and the even order number being listed in sequence table, such as hybridize under strict conditions, or have with the nucleic acid sequence at least about 50% homology, preferably there is at least about 60% homology, more preferably have at least about 70%, 80% or 90% homology even more preferably has at least about 95%, 96%, 97%, 98% or 99% or higher homology.The preferred form of pts protein equally has one or more kinds of bioactivity described herein, and preferred.

PTS polypeptide or its biologically-active moiety can effectively be connected on non-PTS polypeptide and form fused protein.In preferred embodiments, which has the activity different from independent pts protein itself.In other preferred embodiment, which causes required Corynebacterium glutamicum fine chemical yield, production and/or the increase of production efficiency.In particularly preferred embodiments, which is integrated into host cell, the production of required compound in adjustable cell.

On the other hand, the present invention provides the methods for screening the adjustable active molecule of pts protein.The molecule is by the way that perhaps substrate interaction is active to adjust pts protein perhaps in conjunction with the gametophyte of pts protein or through the transcription or translation of adjusting PTS nucleic acid molecules of the present invention with protein molecule itself.

Another aspect of the present invention is related to the method for producing fine chemical.This method is related to the cell that culture contains a kind of carrier, the expression of vectors direct PTS nucleic acid molecules of the present invention, to generate fine chemical.In a preferred embodiment, this method also includes in this step, to transfect cell using the carrier that PTS nucleic acid molecules can be instructed to express the step of acquisition containing the carrier cell.In another preferred embodiment, this method also includes the step of recycling fine chemical from culture medium.In an especially preferred embodiment, cell is rod strain perhaps quarter butt strain or selected from those of being listed in Table 3 bacterial strain.

Another aspect of the present invention relates to adjust the method that molecule generates in microorganism.This method includes using the medicament exposing cell for adjusting pts protein activity or PTS expression of nucleic acid, so that the related activity of cell is changed relative to activity when lacking this medicament.In a preferred embodiment, in order to absorb one or more kinds of carbohydrates, cell is adjusted, so that this Institute of Micro-biology needs the yield of fine chemical or generation efficiency to be improved.The active medicament of pts protein is adjusted, can be the medicament of stimulation pts protein activity or PTS expression of nucleic acid.The example for stimulating the medicament of pts protein activity or PTS expression of nucleic acid has imported the nucleic acid of the pts protein of cell including small molecule, active pts protein and coding.The example for inhibiting the medicament of pts protein activity or expression includes small molecule and antisense PTS nucleic acid molecules.

Another aspect of the present invention, the method for being related to adjusting required compound production in cell, including perhaps the saltant type PTS gene into cells gene is perhaps retained on individual plasmid or is integrated into host cell gene group wild type.If be integrated into host cell gene group, this integration can be arbitrary, or occurred by homologous recombination, so that the copy introduced replaces natural gene, the generation of required compound in cell is caused to be adjusted.In a preferred embodiment, which is increased.In another preferred embodiment, described fine chemical is amino acid.In an especially preferred embodiment, described amino acid is L-lysine.

Specific embodiment

The present invention provides PTS nucleic acid and protein molecules, they participate in Corynebacterium glutamicum intake high energy carbon molecules (for example, glucose, fructose, sucrose), and can also participate in one or more of Cellular Signaling Transduction Mediated approach in the microorganism.Molecule of the invention can be used for adjusting the production of fine chemical in microorganism.This adjusting, it may be possible to due to the increase of high energy species level generated needed for intracellular, for example, ATP, GTP and other for push seem the unfavorable biochemical reaction of energy as fine chemical biosynthesis molecule.The adjusting of fine chemical production is also likely to be due to this fact, i.e., the catabolite of many carbohydrates is used as the intermediate or precursor of other biological route of synthesis, the biosynthesis pathway including certain fine chemicals.Additionally, it is known that pts protein participates in certain Cellular Signaling Transduction Mediated approach, they, which may have the metabolic pathway of one or more of fine chemicals, adjusts activity；By utilizing these pts proteins, the biosynthesis pathway of fine chemical can be activated or inhibit its chemical degradation route.Various aspects of the invention are further described as follows.

I. fine chemical

" fine chemical " this word is well known in the art, the molecule used in various industries including organism generation, such as but is not limited solely to, pharmacy, agricultural and cosmetic industry.This compound includes organic acid, such as tartaric acid, itaconic acid and diaminopimelic acid, protein source and nonprotein source amino acid, purine bases and pyrimidine bases, nucleosides, and nucleotide (is such as description in Kuninaka, A. (1996) Nucleotides and related compounds, p.561-612, in Biotechnology vol.6, Rehm et al., in eds.VCH:Weinheim and its contained bibliography), lipid, saturation and unsaturated fatty acid (such as arachidonic acid), glycol (such as, propane diol and butanediol), aromatic compound (such as, aromatic amine, vanillic aldehyde and indigo), vitamin and Co-factor (referring to Ullmann ' s Encyclopedia of IndustrialChemistry, vol.A27, " Vitamins ", p.443-613 (1996) VCH:Weinheim and referencestherein；And Ong, A.S., Niki, E.& Packer, L. (1995) " Nutrition, Lipids, Health; andDisease " Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia, and the Society for Free Radical Research-Asia, held Sept.1-3,1994 at Penang, Malaysia, AO CS Press, (1995)), enzyme, polyketide (ployketides) (Cane et al., (1998) Science 282:63-68), and all chemical substances described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN:0818805086 and its bibliography.The metabolism of these certain fine chemicals and purposes are further described as follows.

A. the metabolism and purposes of amino acid

Amino acid includes the basic structural unit of all proteins, also necessary to all organism normal cell biological functions." amino acid " this word is well known in the art.The amino acid of protein source has 20 kinds, it is the structural unit of protein, it is connected between each other by peptide bond, rather than proteinogenic amino acids (known to have several hundred kinds) are not appeared in protein under normal conditions (referring to Ulmann ' s Encyclopedia of Industrial Chemistry, vol.A2, p.57-97VCH:Weinheim (1985)).Although l-amino acid is usually the unique type in naturally occurring protein, amino acid can be D- L- optical configuration.The biosynthesis of each in 20 kinds of proteinogenic amino acids perhaps degradation pathway all have the characteristics that in prokaryotic cell or eukaryocyte it is respective (for example, see Stryer, L.Biochemistry, 3^rdEdition, pages 578-590 (1988)).Why " required " amino acid (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) is named in this way, it is because the complicated nutritional condition being generally necessary of these amino acid bios synthesis, they can be converted into remaining 11 kinds of " nonessential " amino acid (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine) by simple biosynthesis pathway.Although higher organism has the ability for synthesizing some this amino acid really, in order to which the synthesis of normal protein must supplement essential amino acid from diet.

They are in addition to the function in protein synthesizes, these amino acid are interesting chemical substance for its own, and many of which has various applications in food, feed, chemistry, cosmetics, agricultural and pharmaceutical industries.Lysine is a kind of important amino acid not only for the mankind in terms of nutrition, and for seeming that poultry and the such nonruminant of pig are also important.Glutamic acid is most common flavor additives useful (monosodium glutamate, MSG), and is widely used in entire food industries, as aspartic acid, glycine, cysteine.Glycine, l-methionine and tryptophan are completely used for pharmaceutical industries.Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are all applied in pharmaceutical industries and cosmetic industry.Threonine, tryptophan and D/L- methionine are common feed addictive (Leuchtenberger, W. (1996) Amino aids-technical production and use, p.466-502 in Rehm et al. (eds.) Biocemistry vol.6, chapter 14a, VCH:Weinheim).In addition, these amino acid are also very useful as the precursor that synthesizing amino acid and protein synthesize, such as N-acetylcystein, S- carboxymethyl-L-cysteine, (S) -5-hydroxyryptophan and other in Ulmann ' s Encyclopedia of Industrial Chemistry, vol.A2, p.57-97 VCH:Weinheim, molecule described in 1985.

In the organism that can generate natural amino acid, such as bacterium, the biosynthesis of these natural amino acids has solved very sufficiently (biosynthesis and its adjusting of bacterial amino acid, referring to Umbarger, H.E. (1978) Ann.Rev.Biochem.47:533-606).Aspartic acid is synthesized by α-ketoglutaric acid reduced form ammonification, and the latter is the intermediate of citrate cycle.Glutamine, proline and arginine are sequentially generated by glutamic acid.The biosynthesis of serine is the process of three steps, starts from 3-phoshoglyceric acid (intermediate of glycolysis), after peroxidation, transamination, each step of hydrolysis, terminates at the amino acid.Cysteine and glycine are all generated by serine；The former is condensed by homocysteine and serine, and the latter is that side chain beta carbon is transferred to what tetrahydrofolic acid obtained, which be catalyzed by serine hydroxymethylase.Phenylalanine and tyrosine are synthesized in the biosynthesis pathway of 9 steps by erythrose-4-phosphate and phosphoenolpyruvate, they are the precursors of glycolytic pathway and pentose phosphate pathway, this two approach are only different after synthesizing prephenic acid.Tryptophan can also be generated by both initial moleculars, but its synthesis is the approach of 11 steps.Tyrosine can also be synthesized by phenylalanine, and reaction is catalyzed by phenylalanine hydroxylase.Alanine, valine and leucine are all the biosynthetic products of glycolysis end product pyruvic acid.Aspartic acid is synthesized by oxaloacetic acid, and the latter is the intermediate of citrate cycle.Asparagine, methionine, threonine and lysine are all transformed by aspartic acid.Isoleucine is formed by threonine.By the approach of a 9 complicated steps, histidine can be generated from a kind of activity sugar, ribose 5-phosphate -1- pyrophosphoric acid.

It cannot be stored beyond amino acid needed for cell protein synthesis, but provide intermediate after being degraded for cell main metabolic pathway and (comment on referring to Stryer, L.Biochemistry 3^rd ed.Ch.21"Amino Acid Degradation and the Urea Cycle"p.495-516(1988)).Although it is useful Metabolic Intermediate that cell, which can convert extra amino acid, many energy, precursor molecule will be consumed and synthesize required enzyme by generating amino acid.Therefore it is not astonishing for the biosynthesis of amino acid being adjusted with feedback inhibition, the presence of special acid can slow down or stop completely the generation (comment for amino acid biosynthetic pathway feedback mechanism of its own, referring to Stryer, L.Biochemistry 3^rd ed.Ch.24"Biosynthesis of Amino Acid and Heme"p.575-600(1988)).Therefore, the yield of any specific amino acids is all limited by intracellular existing amino acid quantity.

B. the metabolism and purposes of vitamin, co-factor and dietetic product

Vitamin, co-factor and dietetic product include another group of molecule, although other biological, such as bacterium, these molecules can be synthesized, higher mammal loses their ability of synthesis and can only absorb.These molecules perhaps itself are the intermediates that bioactive substance or the precursor of the bioactive substance bioactive substance can be electron carrier or various metabolic pathways.In addition to its nutritive value, these compounds also have great industrial value as pigment, antioxidant and catalyst or other processing aids.(the commentary for these compound structures, activity and industrial application, see, for example, Ullmann ' s Encyclopedia of Industrial Chemistry, " Vitamins " vol.A27, p.443-613 VCH:Weinheim 1996.) " vitamin " this word be it is well known in the art, contain needed for organism normal function but again cannot itself synthesis nutrient.Vitamin may include co-factor and dietetic product compound.Non-protein compound needed for term " co-factor " contains the normal enzymatic activity of progress.These compounds can be inorganic or organic；Co-factor molecule of the invention is preferably organic." dietetic product " this word contains the dietary supplement beneficial to plant and animal, especially human body.The example of these molecules is vitamin, antioxidant and certain lipids (such as more saturated fatty acids).

In the organism that can generate these molecules, for example, in bacterium these molecules biosynthesis, most of (Ullman ' s Encyclopedia of Industrial Chemistry that has been accredited, " Vitamins " vol.A27, p.443-613, VCH:Weinheim, 1996；Michal, G. (1999) BiochemicalPathways:An Atlas of Biochemistry and Molecular Biology, John Wiley&Sons；Ong, A.S., Niki, E.& Packer, L. (1995) " Nutrition, Lipids, Health; and Disease " Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia, and the Society for Free Radical Research-Asia, held Sept.1-3,1994 at Penang, Malaysia, AOCS Press:Champaign, IL X, 374 S)

Thiamine (vitamin B1) is by pyrimidine and thiazole through being connected chemically generation.Riboflavin (vitamin B2) is synthesized by 5 '-triphosphoric acid guanosines and 5 '-ribose phosphates.Riboflavin is orderly used to synthesis flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (FAD).The one group of compound (for example, pyridoxol, pyridoxamine, pyridoxime 5'-phosphate, and pyridoxal hydrochloride of commercialization) for being collectively referred to as " vitamin B6 " is all the derivative of common structural unit 5- hydroxyl -6- picoline.Pantothenate (pantothenic acid, (R)-(+)-N- (2,4- dihydroxy -3,3- dimethyl -1- oxo butyl)-Beta-alanine) can be obtained by chemical synthesis or fermentation.The final step of pantothenate biosynthesis includes the condensation of the Beta-alanine and pantoic acid of ATP driving.It is responsible for being converted to pantoic acid and Beta-alanine enzyme, and it is all known for being condensed into the enzyme of pantothenate.The metabolic activity form of pantothenate is coacetylase, and biosynthetic process is 5 enzymatic steps.Pantothenate, pyridoxime 5'-phosphate, cysteine and ATP are the precursors of coacetylase.These enzymes are not only catalyzed the formation of pantothenate, are also catalyzed (R)-pantoic acid, (R)-pantolacton, the generation of (R)-panthenol (dexpanthenol) pantetheine (and its derivative).

Study very detailed by the biosynthesis of precursor molecule heptanedioyl coacetylase to biotin in microorganism, and related several genes have been accredited.Many corresponding protein are also found to take part in the synthesis of iron cluster (Fe-cluster), and are nifS family protein members.Lipoic acid is used as coenzyme from octanoic acid in energetic supersession, can become a part of pyruvate dehydrogenase complex and ketoglurate dehydrogenase compound.Folate is the derivative of one group of folic acid, successively comes from Pidolidone, p-aminobenzoic acid and 6- methylpterin.The biosynthesis for originating in the folic acid and its derivative of Metabolic Intermediate 5 '-triphosphoric acid guanine (GTP), Pidolidone and p-aminobenzoic acid, there is detailed research in certain micro-organisms.

Corrinoid (such as cobalamin, and especially vitamin B12) and porphyrin belong to the chemical substance characterized by tetrapyrrole ring system.The biosynthesis of vitamin B12 is such complexity, so that its feature is thoroughly understood not yet, but many enzymes being related to and substrate are currently known.Niacin (nicotinate) and nicotine are pyridine bottom derivative, also referred to as " niacin usp ".Niacin usp is important coenzyme NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide-adenine dinucleotide phosphate) and its precursor for restoring form.

Although some such compounds can also be produced with Large-scale microbial culture, such as riboflavin and vitamin B6, pantothenic acid and biotin, these compounds are mass produced and largely also rely on acellular chemical system.Only vitamin B12 can only use fermenting and producing due to the complexity of its synthesis.In-vitro method needs considerable substance and time investment, often spends very big.

C. the metabolism and purposes of purine, pyrimidine, nucleosides and nucleotide

Purine and pyrimidine metabolic gene and its corresponding protein are tumor disease therapeutic and the important object for the treatment of of viral infections.Term " purine " and " pyrimidine " contain the nitrogenous base as nucleic acid, coenzyme and nucleotide composition.Term " nucleotide " includes nucleic acid molecules basic structural unit, and by nitrogenous base, pentose, (for RNA, which is ribose to nucleic acid molecules；For DNA, which is deoxyribose) and phosphoric acid composition.Term " nucleosides " contains the molecule as nucleotide precursor, but is the absence of phosphate portion possessed by nucleotide.It by inhibiting the biosynthesis of these molecules, or is suppressed to synthetic nucleic acid molecule and the movement that carries out, the synthesis of RNA and DNA may be inhibited；Inhibit the activity by way of orienting tumour cell, the energy of tumour cell division and duplication may be inhibited.In addition, some nucleotide is not used in form nucleic acid, and it is used as energy storage (such as AMP) or coenzyme (such as FAD and NAD).

Some publications describe the metabolism by influencing purine and/or pyrimidine, these chemical substances as these Medical indications use (such as, Christopherson, R.I.and Lyons, S.D. (1990) " Potent inhibitors of de novo pyrimidine and purine biosynthesis aschemotherapeutic agents. " Med.Res.Reviews 10:505-548).It is related to the research of purine and pyrimidine metabolic enzyme, it concentrates on above the new drug development that can be used, such as, as immunosuppressor or anti-proliferative agent (Smith, J.L., (1995) " Enzyme in nucleotidesynthesis. " Curr.Opin.Struct.Biol.5:752-757；(1995) Biochem Soc.Transact.23:877-902).However, purine and pyrimidine bases, nucleosides and nucleotide also have the function of other: as many fine chemical biosynthesis intermediate (such as, thiamine, S-adenosylmethionine, folic acid, or riboflavin), as cellular energy carrier (such as ATP or GTP), and as chemical substance itself, be typically used as flavour enhancer (such as IMP or GMP) or several medical applications (referring to, such as, Kuninaka, A. (1996) Nucleotidesand Related Compounds in Biotechnology vol.6, Rehm et al., eds.VCH:Weinheim, , p.561-612).Equally, it is related to the enzyme of purine, pyrimidine, nucleosides or nucleotide metabolism, increasingly becomes the effect target for the chemical substance for being used as protection crops developed, these chemical substances include fungicide, herbicide and insecticide.

In bacterium the metabolism of these compounds have feature (comment referring to, such as Zalkin, H.andDixon, J.E. (1992) " de novo purine nucleotide biosynthesis ", in:Progress in NucleicAcid Research and Molecular Biology, vol.42, Academic Press:, p.259-287；AndMichal, G. (1999) " Nucleotides and Nucleosides ", 8 in:BiochemicalPathways:An Atlas of Biochemistry and Molecular Biology, Wiley:New York of Chapter).Purine metabolism is always emphasis research topic, and it is necessary to cell normal function.The purine metabolism being damaged in higher mammal can result in serious disease, such as gout.Purine nucleotides is synthesized by 5 '-ribose phosphates, pass through series of steps, by intermediate 5 '-phosphoric acid inosine (IMP), it is final to generate 5 '-monophosphate guanines (GMP) and 5 '-monophosphate adenines (AMP), and the triphosphate forms as nucleotide are formed by them.These compounds also serve as energy storage, are degraded to a variety of different biochemical processes in cell and provide energy.The biosynthesis of pyrimidine is by forming 5 '-phosphoric acid uridines (UMP) by 5 '-ribose phosphates.Next UMP is transformed into 5 '-triphosphoric acid cytimidines (CTP).The deoxy forms of all these nucleotide are generated by a step reduction reaction, by nucleotide diphosphonic acid ribose form to nucleotide diphosphonic acid deoxyribose form.One is phosphorylated, these molecules can participate in the synthesis of DNA.

D. the metabolism and purposes of trehalose

Trehalose includes two glucose molecules, is connected by α, α -1,1.Usually it is used as sweetener in food industries, in dried foods or frozen food additive and beverage.Moreover, it is also employed in pharmacy, cosmetics and biotechnology industry (see, e.g. Nishimoto et al., (1998) U.S.Patent No.5,759,610；Singer, M.A.and Lindquist, S. (1998) Trends Biotech.16:460-467；Paiva, C.L.A.and Panek, A.D. (1996) Biotech.Ann.Rev.2:293-314；And Shiosaka, M. (1997) J.Japan 172:97-102).Enzyme in many microorganisms can produce trehalose, and it is naturally discharged into surrounding media, and the technically well known method that can be used therefrom is collected.

II. phosphoenolpyruvate: sugar phosphotransferase system

Cell fast-growth in the medium and division are largely dependent upon cellular uptake and the degree using high energy species, such as glucose or other carbohydrates.There are different transport proteins, they transport different carbohydrates in cell.There are carbohydrate transport protein matter, such as transhipment glucose, fructose, mannose, galactolipin, ribose, sorbose, ribulose, lactose, maltose, sucrose or gossypose, also there is the transport protein matter of starch and cellulose degradation product.Other movement systems are responsible for inputting alcohol (such as methanol or ethyl alcohol), alkane, fatty acid, and the organic acid as acetic acid or lactic acid.In bacterium, by various mechanism, carbohydrate can be transported into cell through cell membrane.Other than with the cotransporting of proton, most-often used carbohydrate capture process first is that phosphoenolpyruvate: sugar phosphotransferase system (PTS).The system is not only catalyzed the transhipment (with phosphorylation) of carbohydrate and hexitol, but also adjusts the cell metabolism for being adapted to carbohydrate validity.The PTS system exists only in bacterium, and is not present in archeobacteria and eukaryocyte.

In terms of function, PTS system includes two kinds of cytoplasm proteins, and (every kind is all referred to as and specifically descends target " enzyme II ", such as " enzyme II with carbohydrate for the special integration of the carbohydrate of enzyme I and HPr and indefinite number and peripheral membrane protein transhipment complex^Glu" indicate to combine the enzyme II complex of glucose).It is known to monosaccharide, disaccharides or the special enzyme II of trisaccharide, seem to glucose, fructose, mannose, galactolipin, ribose, sorbose, ribulose, lactose, maltose, sucrose or gossypose.Phosphate group is transferred to phosphocarrier protein HPr from phosphoenolpyruvate (PEP) by enzyme I.Then phosphate group is transferred to different enzyme II again and transports complex by HPr.Although the amino acid sequence of enzyme I and HPr be in all bacteriums it is very similar, PTS transfer body can be divided into the incoherent several families of structure.In addition, the number and homology of these genes are also different in different bacteriums.Genome of E.coli encodes 38 different pts proteins, wherein 33 are the subunits for belonging to 22 different transfer bodies.Mycoplasma genitalium (M.genitalium) genome contains enzyme I and HPr gene each one, and only there are two PTS to shift body gene.T.palladium and chlamydia trachomatis (C.trachomatis) contain enzyme I and HPr similar protein gene, but shift body gene without PTS.

All PTS transfer bodies include 3 functional units, IIA, IIB and IIC, their protein subunits either in complex are (for example, IIA^GlcIICB^Glc) either single polypeptide chain structural region (for example, IICBA^GlcNAc).IIA and IIB is successively transmitted to phosphate group from HPr the carbohydrate being transported.IIC contains carbohydrate-binding sites, and crosses over inner membrance 6 or 8 times.The transient phosphorylation in carbohydrate transfer and the region IIB is coupling.In its histidine residues phosphorylation occurs for enzyme I, HPr and IIA, and IIB subunit is that phosphorylation occurs in histidine residues or cysteine residues, this depends on related specific transporter.The phosphorylation of carbohydrate, has the advantage that after input, it can prevents carbohydrate from spreading cell membrane and return in culture medium, because electrically charged phosphate group cannot pass through the hydrophobic core of cell membrane.

Some pts proteins also play a significant role in signal transduction in the cell other than active carbohydrate transport function.These subunits adjust their purpose object by allosteric or phosphorylation.Their adjusting activity changes with phosphorylation degree (for example, ratio of unphosphorylated form and phosphorylation form), and the latter changes as sugar dependence dephosphorylation and phosphoenolpyruvate rely on the ratio of phosphorylation again.The example of this pts protein adjusted into the cell includes passing through IIA in Escherichia coli^GlcDephosphorylation inhibits glycerokinase, and passes through its phosphorylation form activated adenyl cyclase.Moreover, in these microorganisms some transporters the region HPr and IIB, gene expression is adjusted by the reversible phosphorylation of transcription antitermination.In gram-positive bacterium, the activity of HPr is by HPr special serine kinase and phosphoric acid enzyme adjustment.For example, the HPr of -46 phosphorylation of serine, function is the co-repressor as the sub- CcpA of Transcription inhibition.Finally, it was found that unphosphorylated enzyme I inhibits the sensor kinase c heA in bacterium chemotactic device, the kinases provides directly connection (Sonenshein between bacterial saccharides binding transport system and the system of control bacteria motility, A.L., etal., eds.Bacillus subtilis and other gram-positive bacteria.ASM:Washington, D.C.；Neidhardt, F.C., et al., eds. (1996) Escherichia coli and Salmonella.ASM Press:Washington, D.C；Lengeler et al., (1999) .Biology of Prokaryotes.Section II, pp.68-87, Thieme Verlag:Stuttgart)

III. element of the invention and method

The present invention is at least partly built upon on the basis of discovery recruit, PTS nucleic acid and protein molecule are called it as herein, they participate in Corynebacterium glutamicums to high energy carbon molecules (such as, glucose, fructose, sucrose) intake, and one or more of Cellular Signaling Transduction Mediated approach in these microorganisms can also be participated in.In one embodiment, PTS molecule exercises the function of high energy carbon molecules being transferred to cell, the biochemical reaction energy supply that energy provided by these molecular degradations can be unfavorable for energy in cell, moreover, their catabolite may be used as the intermediate or precursor of a lot of other metabolic pathways.In another embodiment, PTS molecule can participate in one or more of Cellular Signaling Transduction Mediated approach, wherein the presence of PTS molecular modification form (for example, phosphorylation PTS molecule), signal transduction cascade reaction can be participated in, which adjusts one or more cell processes.In a preferred embodiment, the activity of PTS molecule of the present invention, has an impact for the fine chemical needed for the micro-organisms.In an especially preferred embodiment, the activity of PTS molecule of the present invention is regulated so that yield, production and the production efficiency of one or more kinds of fine chemicals in Corynebacterium glutamicum are also adjusted.

Term " pts protein " or " PTS polypeptide " contain those participate in from extracellular medium to cell interior absorb one or more kinds of energy-rich compounds (such as, monosaccharide, disaccharides or oligosaccharides seem fructose, mannose, sucrose, glucose, gossypose, galactolipin, ribose, lactose, maltose and sorbose) protein.These pts proteins can also participate in one or more of Cellular Signaling Transduction Mediated approach, such as but be not limited solely to, those control different carbohydrates and absorb approach into cell.The example of pts protein includes the protein that those are encoded by the PTS gene for being listed in Serial No. odd number in sequence table.About the comprehensive reference document of PTS system, referring to: Stryer, L. (1998) Biochemistry, Chapter 37: " Membrane Transport ", W.H.Freeman:New York, p.959-961；Darnell, J.et al. (1990) Molecular Cell BiologyScientific American Books:New York, p.552-553, and Michal, G., ed. (1999) Biochemical Pathway；An Atlas of Biochemistry and Molecular Biology, Chapter 15 " Special Bacterial Metabolism ".Term " PTS gene " or " PTS nucleic acid sequence " contain the nucleic acid sequence of coding pts protein, and the latter includes 5 ' and 3 ' sequence areas of coding region and corresponding untranslated.The example of PTS gene includes the gene that those are listed in Table 1.Term " production " or " productivity " be it is well known in the art, contain within given time and given fermentation volume, the concentration of tunning (for example, required fine chemical) (for example, product kg per liter per hour).Term " production efficiency " contains, the time needed for reaching specific production level (for example, it is desired to how long just cell can be made to reach specific fine chemical).Term " income " " product/carbon income " be it is well known in the art, contain the efficiency for carbon source being converted to product (such as fine chemical).For example, often writing every kilogram of carbon source of product kg.By improving the income or production of compound, the quantity of recycling molecule can be increased, or increase the quantity of the useful recycling molecule of the compound in the culture for giving quantity within given time.Term " biosynthesis " or " biosynthesis pathway " be it is well known in the art, contain in cell, from intermediate compound by may be multistep and be highly regulated process, synthesize compound, especially organic compound.Term " degradation " or one " degradation pathway " are well known in the art, it contains in cell, by may be multistep and be highly regulated process, compound, preferably organic compound, it is decomposed into catabolite (in general, being smaller or the smaller molecule of complexity).Term " metabolism " be it is well known in the art, contain the whole of the biochemical reaction occurred in organism.Thus, the metabolism (for example, seeming amino acid metabolism as glycine) of special compound includes all biological synthesis relevant to the compound, modification and degradation pathway in cell.Term " transhipment " and " input " be it is well known in the art, contain one or more kinds of compounds across cell membrane easyization movement, and these compounds pass through cannot pass through cell membrane otherwise.

In another embodiment, PTS molecule of the invention can be adjusted in microorganism, such as in Corynebacterium glutamicum, the generation of required compound such as fine chemical.One or more kinds of PTS molecules of the invention can be operated using recombinant genetic technology, to adjust its activity.For example, the PTS system that a kind of protein for participating in the glucose input that PTS is mediated can be adjusted, and make its activity optimization, and input glucose, can transport greater number of glucose into cell.Because glucose molecule serves not only as energy to push the unfavorable biochemical reaction of energy, such as fine chemical biosynthesis, and it can be used as the precursor and intermediate of many biological fine chemical biosynthesis pathways (for example, synthesizing serine from 3-phoshoglyceric acid).In each example, the total output or production efficiency that can perhaps increase these required fine chemicals by compound supply needed for increasing production generation are perhaps provided by energy needed for increasing production generation.

In addition, many known pts proteins play a crucial role in signal transduction path in the cell, these approach adjust the cell metabolism for maintaining carbon source supply and carbohydrate intake.For example, as it is known that the increase of intracellular fructose 1,6-diphosphate (compound generated in glycolysis) level, can lead to the phosphorylation of HPr serine residue, to prevent the protein in any PTS carbohydrate transport process as phosphate group donor.Prevent its serine residue from being phosphorylated by mutagenesis HPr, it can be with the activation HPr of composition, to increase the carbohydrate in transporte to cells, may then ensure that intracellular for more energy storages and intermediate/precursor molecule needed for fine chemical biosynthesis needed for one or more.

The nucleic acid sequence of the present invention of separation, included in the genome of Corynebacterium glutamicum strain, which can be obtained by American type culture collection, deposit number ATCC 13032.Isolated Corynebacterium glutamicum PTS DNA nucleic acid sequence, and the Corynebacterium glutamicum pts protein amino acid sequence of prediction, are listed respectively with odd serial numbers and even order number in sequence table.

It has carried out computer analysis, and these nucleic acid sequences has been classified and/or be accredited as the sequence of encoding metabolic pathway proteins matter.

The present invention is also related with such protein, and the amino acid sequence of the protein and amino acid sequence of the invention have sufficient homology the sequence of even order number (for example, in sequence table).As used herein, have has the protein of the amino acid sequence of abundant homology to have at least about 50% homology with the amino acid sequence picked out, such as the overall amino acid sequence picked out with the amino acid sequence picked out.Protein with the amino acid sequence for having very big homology with the amino acid sequence picked out, there can also be at least about 50-60% with the amino acid sequence picked out, preferably there is at least about 60% homology, more preferably there is at least about 70%, 80%, 90% homology, most preferably have at least about 95%, 96%, 97%, 98%, 99% or higher homology.

Pts protein of the invention or its biologically-active moiety or its segment, can participate in transhipment seems that high energy carbon-containing molecules as glucose enter Corynebacterium glutamicum, perhaps the Cellular Signaling Transduction Mediated in the microorganism is participated in or with the one or more activity listed in table 1.

Various aspects of the invention are described in more detail in following each section:

A. the nucleic acid molecules separated

One aspect of the present invention is related to the coding PTS polypeptide of separation or the nucleic acid molecules of its biologically-active moiety, and be used as enough hybridization probe perhaps these segments of the nucleic acid molecule fragment of primer for identifying or the nucleic acid of amplification coding PTS (such as PTS DNA).As used herein, term " nucleic acid molecules " is meant comprising DNA molecular (for example, cDNA perhaps genomic DNA) and RNA molecule (such as mRNA) and by DNA the RNA analog of nucleotide analog generation.The term also includes the non-translated sequence positioned at the end of gene coding region 3 ' and 5 ': at least 20 nucleotide of 3 ' the end downstream sequence of at least 100 nucleotide and gene coding region of 5 ' end upstream sequence of coding region.Nucleic acid molecules can be it is single-stranded or double-strand, it is preferred that being double-stranded DNA." separation " nucleic acid molecules refer to those nucleic acid molecules being separated from each other with other nucleic acid molecules being present in nucleic acid natural origin.It is preferred that " separation " nucleic acid is without containing the natural sequence (for example, the sequence for being located at the end of nucleic acid 5 ' and 3 ') positioned at organism genomic DNA amplifying nucleic acid two sides, nucleic acid is exactly to obtain from the organism.Such as, in various embodiments, isolated PTS nucleic acid molecules can be containing less than about 5kb, 4kb, 3kb, 2kb, the nucleotide sequence of 1kb, 0.5kb or 0.1kb, the sequence are naturally located at the two sides of cell genomic dna nucleic acid molecules, nucleic acid is exactly to obtain from these cells (for example, Corynebacterium glutamicum cell).In addition, " separation " nucleic acid molecules, such as DNA molecular, when producing with recombinant technique precursor or other chemical substances can be free of when perhaps culture medium is when chemical synthesis essentially free of other cellular materials.

Nucleic acid molecules of the present invention, such as the nucleotide sequence of odd serial numbers or its part in sequence table, can be isolated by standard molecular biological technique and sequence information provided herein.Such as, Corynebacterium glutamicum PTS DNA can be from Corynebacterium glutamicum library, the whole for using in sequence table in odd serial numbers sequence a sequence or its part are as hybridization probe, and standard hybridisation methods are (for example, seem description in Sambrook, J., Fritsh, E.F., and Maniatis, T.Molecular Cloning:A Laboratory Manual.2^nd, in ed.Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and isolated.In addition, comprising a nucleic acid sequence of the present invention (such as, the nucleotide sequence of odd serial numbers in sequence table) all or part nucleic acid molecules, polymerase chain reaction can be passed through, use the oligonucleotide primer based on the sequence design, isolated (such as, comprising a nucleic acid sequence of the present invention (such as, the nucleotide sequence of odd serial numbers in sequence table) all or part nucleic acid molecules, polymerase chain reaction can be passed through, it is isolated using the oligonucleotide primer based on the identical sequence design).Such as, mRNA can it is isolated from normal endothelial cell (such as, use the guanidine thiocyanate extracting method in Chirgwin et al. (1979) Biochemistry 18:5294-5299), DNA can by reverse transcriptase (such as, Gibco/BRL, the Moloney MLV reverse transcriptase that Bethesda, MD are provided；Or SeikagakuAmerica, Inc., the AMV reverse transcriptase that St.Peterburg, FL are provided) preparation.It, can be based on the nucleotide sequence design listed in sequence table for the oligonucleotide primer of polymerase chain reaction synthesis.Nucleic acid of the invention, can be used cDNA or genomic DNA as template alternatively, and suitable oligonucleotide primer is expanded according to standard PCR amplification technology.The nucleic acid amplified in this way can be cloned into suitable carrier, and distinguish its feature with DNA sequence analysis.In addition, oligonucleotide corresponding with PTS nucleotide sequence, can be prepared, such as use automatic dna synthesizer with Standard synthetic techniques.

In a preferred embodiment, the nucleic acid molecules of the present invention of separation include the nucleotide sequence listed in sequence table.Nucleic acid sequence of the invention is consistent as those of listing in sequence table with Corynebacterium glutamicum PTS DNA of the invention.These DNA include the sequence (i.e. " coding region " of coding pts protein, it is shown in odd serial numbers in every sequence table: in sequence), and 5 ' non-coding sequence and 3 ' non-coding sequences, it is also shown in odd serial numbers in every sequence table: in sequence.Alternatively, nucleic acid molecules can only include the coding region of sequence table more control sequences.

For the purpose of this application, it is possible to understand that the every nucleic acid and amino acid sequence listed in sequence table have a RXA for identification, RXN, RXS or RXC coding, indicates " RXA ", " RXN ", " RXS ", or " RXC " has 5 numbers (that is, RXA01503, RXN01299 below, RXS00315 or RXC00953).Every nucleic acid sequence contains up to three parts: 5 ' upstream regions, coding region, downstream area.Trizonal each part is all obscured with identical RXA, RXN, RXS or RXC number determination with eliminating.Then narration " sequence of the odd number coding in sequence table " refers to that any nucleic acid sequence in sequence table, these sequences can also mutually be distinguished with their different RXA, RXN, RXS or RXC numbers.The coding region of every this sequence is all translated into corresponding amino acid sequence, these sequences are also listed in sequence table, to follow even order number after corresponding nucleic sequence closely:.For example, the coding region of RXA02229 is listed in SEQ ID NO:1, and the amino acid sequence that it is encoded is listed in SEQ ID NO:2.Sequence of nucleic acid molecules of the invention, the amino acid molecular encoded with it, with identical RXA, RXN, RXS or RXC number are indicated, so that they are easy to connect each other.For example, being appointed as RXA01503, the amino acid sequence of RXN01299, RXS00315 and RXC00953 are RXA01503, RXN01299, the translation in the nucleotide sequence coded region of RXS00315 and RXC00953 nucleic acid molecules respectively.RXA of the present invention, RXN, correspondence and their appointed sequence numbers between RXS and RXC nucleotide and amino acid sequence are listed in Table 1.For example, seem the nucleotide sequence RXN01299 being listed in Table 1 being SEQ ID NO:7, corresponding amino acid sequence is SEQ ID NO:8.

Several genes of the invention are " genes that F- is indicated ".The gene that F- is indicated includes the gene that those are listed in Table 1 and have " F " before RXA, RXN, RXS or RXC are indicated.For example, SEQ ID NO:3, as being indicated in table 1, it is designated as " F RXA00315 ", the gene that an exactly F- is indicated, same there are also SEQ ID NO:9,11 and 13 (are marked as " F RXA01229 ", F RXA01883 in table 1 respectively " and " F RXA01889 ").

In one embodiment, nucleic acid molecules of the invention, which do not include, those of is compiled in table 2 Corynebacterium glutamicum molecule.For dapD gene, the sequence of the gene is published in Wehrmann, A., et al. (1998) J.Bacteriol.180 (12): 3159-3165.However, the present application person it is obtained it is longer than the version delivered very much.It has been said that the version delivered has used the codon in fact of mistake, and therefore only it is demonstrated by a part of true coding region.

In another preferred embodiment, the nucleic acid molecules of the invention of separation are the nucleic acid molecules of nucleotide sequence of the present invention (for example, odd serial numbers in sequence table: sequence) or the complementary molecule of its part comprising those.The nucleic acid molecules complementary with a nucleotide sequence of the invention, refer to listed in the molecule and sequence table a nucleotide sequence (such as, odd serial numbers: sequence) it is sufficiently complementary, therefore it can with a nucleotide sequence hybridization of the invention, to form stable double helix.

Equally in another preferred embodiment, the nucleic acid molecules of the invention of separation, include such nucleotide sequence, the sequence and nucleotide sequence of the invention (such as, odd serial numbers in sequence table: sequence) or its part, have at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% homology, preferably have at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% homology, more preferably have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8 7%, 88%, 88%, 89% perhaps 90% or 91%, 92%, 93%, 94%, and even more preferably have at least about 95%, 96%, 97%, 98%, 99% or higher homology.Range (for example, 70-90% consistency or 80-95% consistency) referenced above intermediate range and consistency value, are also included in the present invention.For example, containing such consistency value range, these ranges are the combinations of the above-cited upper limit and/or lower limit value.In another preferred embodiment, the nucleic acid molecules that the present invention separates include such nucleotide sequence, the sequence can with a nucleotide sequence of the invention (such as, odd serial numbers in sequence table: sequence) or its part hybridized, for example, hybridizing under strict conditions.

In addition, nucleic acid molecules of the present invention only may include a part of odd serial numbers sequences encoding regions in sequence table, for example, may be used as the probe perhaps segment of primer or the segment of coding pts protein biologically-active moiety.The nucleotide sequence gone out by Corynebacterium glutamicum PTS gene cloning, allow to generate probe and primer, the design of these probes and primer is for identifying and/or cloning the PTS homologue in PTS homologue and other bar bacteriums or affinity species of other cell types perhaps in other organisms.Probe/primer is typically include the oligonucleotide quite purified.Oligonucleotide is typically include the region of such one section of nucleotide sequence, the region is under stringent hybridization conditions, with nucleotide sequence of the present invention (such as, odd serial numbers sequence in sequence table) sense strand, at least about 12 of the antisense sequences of these sequences or its naturally occurring mutant, preferably about 25, preferred about 40,50 or 75 continuous nucleotides hybridization.Based on the primer of nucleotide sequence of the present invention, it can be used for cloning the PCR reaction of PTS homologue.Based on the probe of PTS nucleotide sequence, it can be used for detecting identical or homologous protein transcription or genome sequence.In a preferred embodiment, probe includes even more other attachment labelling groups, such as labelling groups can be the co-factor of radioactive isotope, fluorescent chemicals, enzyme or enzyme.This probe may be used as a part of diagnostic test kits, the kit is used to identify the cell of false demonstration pts protein, it seem the level by PTS code nucleic acid in measurement sample cell, such as, the level of PTS mRNA is detected, either measures whether genome PTS gene is mutated or is lacked.

In one embodiment, a kind of protein of nucleic acid molecule encoding of the present invention or its part, the protein or the amino acid sequence of its part and amino acid sequence of the invention (such as, even order sequence in sequence table) there is sufficient homology, so that the protein or its part have the ability high energy carbon molecules (such as glucose) to input Corynebacterium glutamicum, one or more of Cellular Signaling Transduction Mediated approach can also be participated in.As used herein, term " sufficient homology " refers to protein or the amino acid sequence of its part, containing minimal amount with it is consensus amino acid sequence of the present invention or of equal value (such as, with the amino acid residue with the amino acid residue similar side chain in sequence table even order sequence) amino acid residue, so that the protein or its part, the one or more of Cellular Signaling Transduction Mediated approach in the microorganism can also be participated in seeming the input Corynebacterium glutamicum of high energy carbon molecules as glucose.The Protein members of this metabolic pathway, as described herein, function is that handle seems the input Corynebacterium glutamicum of high energy carbon molecules as glucose, can also participate in the one or more of Cellular Signaling Transduction Mediated approach in the microorganism.This active example is also described.Thus, " function of pts protein " contributes the repertoire of the one or more of carbohydrate transporting pathway based on phosphoenolpyruvate and/or adjusting, and/or directly or indirectly contributes to the yield of one or more kinds of fine chemicals, production and/or production efficiency.The active example of pts protein is listed in table 1.

In another embodiment, protein and whole amino acid sequences of the invention have at least about homology of 50-60%, preferably there is at least about homology of 60-70%, more preferably there is at least about 70-80%, 80-90%, the homology of 90-95%, most preferably have at least about 96%, 97%, 98%, 99% or higher homology (for example, even order number in sequence table: sequence).

The part of PTS nucleic acid molecule encoding protein of the present invention, the preferably biologically-active moiety of pts protein.As used herein, term " biologically-active moiety of pts protein " is meant comprising part as pts protein, such as structural domain/primitive, the part or can participate in the one or more of Cellular Signaling Transduction Mediated approach in the microorganism seeming the input Corynebacterium glutamicum of high energy carbon molecules as glucose.It can carry out a kind of enzyme activity assay, to determine perhaps whether its biologically-active moiety takes part in seeming the input Corynebacterium glutamicum of high energy carbon molecules as glucose or take part in one or more of Cellular Signaling Transduction Mediated approach in the microorganism pts protein.This analysis method for be familiar with for ordinary skill be it is well known, be described later in detail in the example 8 of example.

Encode the additional nucleic acid fragment of pts protein biologically-active moiety, it can be prepared by the following method, separation amino acid sequence of the present invention (such as, even order number in sequence table: sequence) a part, express pts protein or polypeptide coded portion (such as, pass through in-vitro recombination expression), and estimate the activity of pts protein or peptide coding part.

Because of the degeneracy of genetic codon, and it is possible thereby to encode to obtain pts protein identical with nucleotide sequence encoding protein matter of the present invention, so the present invention further includes the nucleic acid molecules different from nucleotide sequence of the present invention (for example, odd serial numbers in sequence table: sequence) (and its part).In another embodiment, the nucleic acid molecules of the invention of separation have such nucleotide sequence, which has the protein for the amino acid sequence (for example, even order number :) listed in sequence table.Equally in another embodiment, the Corynebacterium glutamicum protein of nucleic acid molecule encoding overall length of the present invention, the protein and amino acid sequence of the invention (by odd serial numbers in sequence table: open reading frame encodes) have sufficient homology.

In one embodiment, sequence of the invention is not meant to include sequence known in the art in the past, for example, those be listed in table 2 or table 4 before the present invention with regard to effective Genbank sequence, this will be understood by for ordinary skill for being familiar with.In one embodiment, the present invention includes such nucleotide sequence and amino acid sequence, the sequence and nucleotide sequence of the invention and amino acid sequence have the consistency of certain percentage, the percentage is greater than the percentage of sequence (for example, the Genbank sequence (or protein of the sequential coding) listed in table 2 or table 4) and nucleotide sequence and Amino acid sequence identity of the invention known in the art.Such as, the present invention includes to show to be greater than and/or the nucleotide sequence of at least 44% consistency with the nucleotides sequence for being marked as RXA01503 (SEQ ID NO:5), it shows and is greater than and/or the nucleotide sequence of at least 41% consistency with the nucleotides sequence for being marked as RXA00951 (SEQ ID NO:15), and show with the nucleotides sequence for being marked as RXA01300 (SEQ ID NO:21) and to be greater than and/or the nucleotide sequence of at least 38% consistency.It is familiar with ordinary skill, Percent Identity is calculated by the GPA- met for 3 highests that each particular sequence provides listed in inspection table 4, and Percent Identity is calculated by subtracting highest GPA- from 100 percent, the low side thresholding of any particular sequence Percent Identity of the present invention can be calculated.Being familiar with ordinary skill may also be appreciated that, its Percent Identity be greater than so calculated low side thresholding (such as, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferred at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher consistency) nucleic acid and amino acid sequence, be also included in the present invention.

Being familiar with ordinary skill can be appreciated that, other than the Corynebacterium glutamicum PTS nucleotide sequence listed in sequence table with odd serial numbers, the DNA polymorphism for causing pts protein amino acid sequence to change can exist in certain group (such as Corynebacterium glutamicum group).The genetic polymorphism of this PTS gene, can due to natural conditions variation and exist in the Different Individual of a group.As used herein, term " gene " and " recombination " refer to that preferred pts protein is Corynebacterium glutamicum pts protein containing the nucleic acid molecules for the open reading frame for encoding pts protein.The variation of this natural conditions can typically cause the variation of PTS gene nucleotide series 1-5%.It is any and all as caused by the variation of natural conditions, and do not change pts protein functional activity, the polymorphism of the variation of this nucleotide and caused PTS amino acid belongs within the scope of the invention.

The nucleic acid molecules of corresponding natural variant, with the non-glutamic acid bar bacterium homologue of Corynebacterium glutamicum PTS DNA of the present invention, it can the homology based on disclosed herein they and Corynebacterium glutamicum PTS nucleic acid molecules, use Corynebacterium glutamicum DNA or its part as hybridization probe, it is isolated according to standard hybridisation methods under stringent hybridization conditions.Therefore, in another embodiment, the length of the nucleic acid molecules of the present invention of separation at least 15 nucleotide, under strict conditions with odd serial numbers containing ordered list: the making nucleic acid molecular hybridization of nucleotide sequence.In other embodiments, the length of nucleic acid molecules at least 30,50,100,250 or more nucleotide.As used herein, term " hybridizing under strict conditions " means the condition of description such hybridization and cleaning, has the nucleotide sequence of at least 60% homology to keep typically hybridizing between each other each other under this condition.It is preferred that this condition is that have at least about 65% between sequence, more preferably there is at least about 70%, and even more preferably have at least about 75 or higher homology, keeps typical hybridization between each other.This stringent condition for be familiar with ordinary skill be it is known, can be found in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Stringent hybridization condition that is a kind of preferred but not being limitation is hybridized for about 45 DEG C in 6X sodium chloride/sodium citrate (SSC), then uses 0.2X SSC, and 0.1%SDS is primary or repeatedly in 50-65 DEG C of cleaning.It is preferred that the nucleic acid molecules of the invention of separation are equivalent to obtain naturally occurring nucleic acid molecules under stringent hybridization conditions with nucleotide sequence hybridization of the invention.As used herein, " naturally occurring " nucleic acid molecules refer to RNA or DNA molecular with nucleotide sequence present in nature (for example, coding native protein).In one embodiment, nucleic acid encode natural glutamate bar bacterium pts protein.

Being familiar with ordinary skill will be further appreciated that, other than the naturally occurring PTS sequence variants present in the group, change can be introduced into nucleotide sequence of the present invention by mutation, the function so as to cause the change of the amino acid sequence of pts protein encoded, without changing pts protein.For example, can be in nucleotide sequence of the present invention, progress can cause the nucleotide of the amino acid substitution of " nonessential " amino acid residue to replace." nonessential " amino acid residue refers to such residue, the residue can pts protein wild-type sequence (such as, even order number in sequence table: sequence) in change, activity without changing pts protein, and " required " amino acid residue is necessary to pts protein activity.However, other amino acid residues (for example, those non-conservative in PTS active structure domain or only semi-conservative amino acid residues) may be not required activity, therefore can also be changed in the case where not changing the active situation of PTS.

Therefore, another aspect of the present invention is related to encoding the nucleic acid molecules of such pts protein, which contains the variation of the amino acid residue nonessential to PTS activity.The amino acid sequence of these protein is different from even order number in sequence table: sequence, but still keeps at least one PTS activity described herein.In one embodiment, isolated nucleic acid molecules include the nucleotide sequence of one section of coding protein, wherein the amino acid sequence of the protein and amino acid sequence of the invention have at least about 50% homology, and it can be seeming that high-energy carbon-containing molecules as glucose are transported into Corynebacterium glutamicum, perhaps the Cellular Signaling Transduction Mediated in the microorganism is participated in or with the one or more activity listed in table 1.It is preferred that, odd serial numbers amino acid sequence in the protein of nucleic acid molecule encoding and sequence table, there is at least about homology of 50-60%, more preferably has at least about homology of 60-70% with this sequence, even more preferably have at least about 70-80% with this sequence, 80-90%, the homology of 90-95% most preferably has at least about 96%, 97% with amino acid sequence of the invention, 98% or 99% homology.

In order to determine two kinds of amino acid sequences (such as, a kind of amino acid sequence of the invention and its mutant forms) or two kinds of nucleic acid sequences percent homology, the purpose compared for optimum, to sequence carry out alignment (such as, in order to which perhaps the optimum alignment of nucleic acid progress can introduce gap in the sequence of a kind of protein or nucleic acid with other protein).Then the nucleotide of the amino acid residue of more corresponding amino acid position or nucleic acid position.When a sequence (such as, an amino acid sequence of the invention) in a position by with other sequences (such as, the mutant forms of amino acid sequence) the identical amino acid residue in corresponding position or nucleotide is when occupying, the molecule is homologous (that is, " consistency " of amino acid or nucleic acid " homology " and amino acid or nucleic acid is identical as used herein) in this position.Percent homology between two sequences is the function (that is, % consistency=same position #/whole position #x100) that a same position number is divided equally by sequence.

Separation with present protein sequence (such as, even order number in sequence table: sequence) homologous coding pts protein matter nucleic acid molecules, it is generated and being replaced by introducing one or more nucleotide into nucleotide sequence of the present invention, be inserted into, lack, to introduce one or more amino acid substitution, insertion, missing in coding protein.Standard technique, such as the mutagenesis that direct mutagenesis and PCR are mediated can be used, mutation is introduced in nucleotide sequence of the present invention.It is preferred that conservative amino acid substitution is that non-essential amino acid residues expected from one or more carry out." conservative amino acid substitution " refers to amino acid residue replaced the amino acid residue with similar side chain.Amino acid residue families with similar side chain, technically there is regulation.These families include, with basic side chain amino acid (such as, lysine, arginine, histidine), with acid side-chain amino acid (such as, aspartic acid, glutamic acid), with no charge polarity side chain amino acid (such as, glycine, aspartic acid, glutamine, serine, threonine, tyrosine, cysteine), with non-polar sidechain amino acid (such as, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), with β-branched side chains amino acid (such as, threonine, valine, isoleucine), and with fragrance group side chain amino acid (such as, tyrosine, phenylalanine, tryptophan, histidine).Therefore, it is contemplated that pts protein in non-essential amino acid residues, preferably by other amino acid substitutions in same side chain family.In addition, in another embodiment, it can be in PTS coded sequence overall length or part, random introducing mutation, such as by saturation mutagenesis, there is the PTS activity of PTS active mutant, the mutant filtered out according to identification described herein.In a sequence table after odd serial numbers nucleotide sequence mutagenesis, protein encoded can be recombinantly expressed, and protein active can also be with, such as using analysis (referring to the example 8 of example) described herein, is determined.

Other than the nucleic acid molecules of coding pts protein matter described above, another aspect of the present invention is also related with isolated antisense nucleic acid molecule." antisense " nucleic acid includes the nucleotide sequence with " ariyoshi " complementary nucleic acid of coding protein, such as complementary with double chain DNA molecule coding strand or complementary with mRNA sequence.Therefore, antisense nucleic acid can be by hydrogen bond and having phosphorothioate odn connection.Antisense nucleic acid can be complementary with whole PTS coding strands, can also only with its partial complementarity.In one embodiment, antisense nucleic acid molecule, " coding region " antisense with the nucleotide sequence coded chain of coding pts protein.Term " coding region " refers to the nucleotide sequence region (for example, whole coding regions of SEQ ID NO.5 (RAX 01503) include 1 to 249 nucleotide) of the codon comprising translating into amino acid residue.In another embodiment, antisense nucleic acid molecule, the antisense with the nucleotide sequence coded chain of coding PTS.Term " non-coding region " refers to that coding region two sides do not translate into 5 ' and 3 ' sequences (i.e. 5 ' and 3 ' untranslated regions) of amino acid.

In view of the coding strand sequence (for example, the odd serial numbers sequence listed in sequence table) of the coding PTS announced herein, antisense nucleic acid of the present invention can be designed according to the base pairing rules of Watson and Crick.Antisense nucleic acid molecule can be complementary with whole coding regions of PTS mRNA, but more preferably such oligonucleotide, which is only antisense with the part of the coding region of PTS mRNA or non-coding region.For example, antisense oligonucleotide can be complementary with the region near PTS mRNA translation initiation position.For example, the length of antisense oligonucleotide can be 5,10,15,20,25,30,35,40,45 or 50 nucleotide.Program known in the art can be used in antisense nucleic acid molecule of the invention, passes through chemical synthesis or enzymatic connection reaction building.Naturally occurring nucleotide or the various nucleotide by modification can be used, chemical synthesis antisense nucleic acid (such as antisense oligonucleotide), those pass through the nucleotide of modification, it is the biological stability in order to increase molecule, or designed to increase antisense nucleic acid and have the physical stability for forming double helix between phosphorothioate odn, the nucleotide that phosphorothioate analogue and acridine replace can be used for example.The example that can be used for generating the modified nucleotide of antisense nucleic acid includes, 5 FU 5 fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5- (carboxy hydroxy methyl) uracil, 5- carboxymethylamino methyl -2- thiouracil, 5- carboxymethylamino methyluracil, dihydrouracil, beta-D- galactosyl inosine, N6- isopentyl adenine, 1- methyl guanine, 1-methylinosine, 2, 2- dimethylguanine, 2- methyl adenine, 2- methyl guanine, 3- methylcystein, 5-methylcytosine, N6- adenine, 7- methyl guanine, 5- Methylaminomethyl uracil, 5- Methoxyamino methyluracil -2- paper substrate, beta-D- is sweet Reveal glycosyl queosine, 5 '-methoxycarbonyloxymethyl uracils, 5- methoxyuracil, 2- methyl thio-N6- isopentyl adenine, uracil -5- (V) containing fluoroacetic acid, wybutoxosine, pseudouracil, queosine, the thio cytimidine of 2-, 5- methyl -2- paper substrate, 2- paper substrate, 4- paper substrate, methyl uracil, the oxygen-containing methyl acetate of uracil -5-, uracil -5- (v) containing fluoroacetic acid, 5- methyl -2- paper substrate, 3- (3- amino -3-N-2- carboxypropyl) uracil, (acp3) w, and 2, 6- diaminopurine.In addition, expression vector biosynthesis can be used in antisense nucleic acid, amplifying nucleic acid is subcloned into expression vector (that is, by the RNA of insertion transcribed nucleic acid, the purpose nucleic acid relative to insertion is antisense orientation, is partially further discussed below below) by antisense orientation.

Antisense nucleic acid molecule of the invention is typically applied to cell and is perhaps generated in situ so that they and then can inhibit the expression of protein, for example, inhibiting transcription and/or translation with the cell mRNA and/or genomic DNA hybridization or combination of coding pts protein.Hybridization can form stable double helix by the way that common nucleotides are complementary, alternatively, for example, when antisense nucleic acid molecule combination DNA double spiral, the major groove generation Specific Interactions of it and double helix.Antisense molecule can be modified, so that the molecule can be with receptor perhaps in conjunction with the antigentic specificity of specific cells surface expression for example, the perhaps combination of the antibody antibody and cell surface receptor or the antigen binding of antisense nucleic acid molecule and polypeptide.Vehicle delivery described herein also can be used to cell in antisense nucleic acid molecule.Intracellular sufficient concentrations of antisense molecule, such carrier are preferably that is, in the carrier, antisense nucleic acid molecule is placed under the control of protokaryon, virus or eukaryotic promoter in order to obtain.

And in another embodiment, antisense nucleic acid molecule of the invention is a kind of α-anomeric nucleic acid molecule.α-anomeric nucleic acid molecule forms special double-stranded hybrid with complementary RNA, two strands of chains move towards parallel to each other in hybrid, this (Gaultier et al. (1987) Nucleic Acids.Res.15:6625-6641) opposite with common β-unit.Antisense nucleic acid molecule also may include 2 '-o- methyl ribonucleotides (Inoue et al. (1987) Nucleic Acids.Res.15:6131-3148) or chemistry RNA-DNA analog (Inoue et al. (1987) FEBS Lett.215:327-330).

And in another embodiment, antisense nucleic acid molecule of the invention is ribozyme.Ribozyme is catalytic type RNA molecule, has ribonuclease activity, can cutting single-chain nucleic acid, such as mRNA, it has the region complementary with single-chain nucleic acid.Therefore, ribozyme (for example, hammerhead ribozyme (being described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used for catalysis cutting PTS mRNA transcript, to inhibit the translation of PTS mRNA.There is the ribozyme of specificity for PTS coding nucleic acid molecule, can be designed based on the PTS DNA nucleotide sequence (i.e. SEQID NO:5 (RAX 01503)) announced herein.For example, the derivative of tetrahymena L-19 IVS RNA can be constructed, the nucleotide sequence of active site is complementary with the nucleotide sequence of the PTS- coding mRNA cut.See, e.g., Cech et al.U.S.Patent No.4,987,071 and Cech et al.U.S.Patent No.5,116,742.In addition, PTS mRNA, which can be used for screening in RNA molecule library, has the active catalytic type RNA of Specific ribozyme.See, e.g., Bartel, D.and Szostak, J.W. (1993) Science 261:1411-1418.

In addition, by the handle nucleotide sequence complementary with PTS nucleotide sequence adjustment region (for example, PTS promoter and/or enhancer) as target, triple-helix structure is formed, it can inhibit the expression of PTS gene, which can prevent transcription of the PTS gene in aim cell.Referring generally to Helene, C. (1991) Anticancer Drug Des.6 (6): 569-84；Helene, C.etal. (1992) Ann.N.Y.Acad.Sci.660:27-36；And Maher, L.J. (1992) Bioassays 14 (12): 807-15.

B. recombinant expression carrier and host cell

Another aspect of the present invention relates to carriers, preferably the expression vector containing coding pts protein (or its part) nucleic acid.As used herein, term " carrier " is to refer to connect other nucleic acid, and the nucleic acid molecules for transporting it.A type of carrier is " plasmid ", and plasmid refers to circular double-stranded DNA ring, wherein being connected with additional DNA fragmentation.Another type of carrier is viral vectors, wherein additional DNA fragmentation may be coupled in viral genome.Certain carriers can independently be replicated (for example, bacteria carrier and episomal mammalian vectors with bacterial origin of replication) in the host cell that they are introduced into.Other carriers (for example, non-add type mammalian vector) are once being introduced into the genome that host cell will be integrated into host cell, to replicate together with host genome.In addition, certain carriers can instruct the expression of connected gene.These carriers are here referred to as " expression vector ".In short, the expression vector that recombinant DNA technology uses is often plasmid form.In the present note, " plasmid " and " carrier " can be used interchangeably, because plasmid is most-often used carrier format.However, the present invention includes the other forms of these expression vectors, such as viral vectors (for example, replication defect type retrovirus, adenovirus and adeno-associated virus), their functions having the same intentionally.

Recombinant expression carrier of the present invention includes nucleic acid of the invention, the nucleic acid exists in the form of being suitble to expression of nucleic acid in host cell, this means that recombinant expression carrier contains one or more of adjusting sequences, these sequences are selected based on the host cell for being used as expression, they are connected in the nucleic acid sequence to be expressed by feasible.In recombinant expression carrier, " feasible connection " is meant that, it nucleotide sequence interested and adjusts sequence and is attached (for example, in vitro in transcription/translation system, or in the host cell that carrier is introduced into) in a manner of allowing nucleotide sequence to express.Term " adjusting sequence " is meant including promoter, enhancer and other expression control element (for example, polyadenylation signal).This adjusting sequence exists, for example, Goeddel；It is described in GeneExpression Technology:Methods in Enzymology 185, Academic Press, SanDiego, CA (1990).Adjust sequence include, those in many host cell types, instruct nucleotide sequence constitutive expression sequence and those in certain host cells, instruct nucleotide sequence express sequence.The preferred sequence that adjusts is, for example, seeming cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI^q, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny-, SPO2-, λ-P_ROr λ P_LSuch promoter, these promoters preferably use in bacterium.In addition adjusting sequence is, for example, the promoter of yeast and fungi, such as ADC1, MF α, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, the promoter of plant, for example, CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, no or ubiquitin- or phaseolin- promoter.Also artificial promoter can be used.These can be appreciated that the i.e. design of expression vector depends on these factors: the selection of the host cell for conversion, the expression etc. of required protein for being familiar with ordinary skill.Expression vector of the invention can introduce host cell, to generate the protein of encoded by nucleic acid described herein, perhaps polypeptide includes fused protein or polypeptide (for example, the mutant form of pts protein, pts protein, fused protein etc.).

Recombinant expression carrier of the invention can be designed, for expressing pts protein in protokaryon or eukaryocyte.Such as, PTS gene can express in following cell, it seem bacterial cell as Corynebacterium glutamicum, insect cell (uses rhabdovirus expression vector), yeast and other fungal cells are (referring to Romanos, M.A.et al. (1992) " Foreign gene expression in yeast:areview ", Yeast 8:423-488；Van den Hondel, C.A.M.J.J.et al. (1991) " Heterologousgene expression in filamentous fungi " in:More Gene Manipulations in Fungi, J.W.Bennet&L.L.Lasure, eds., p.396-428:Academic Press:San Diego；And van denHondel, C.A.M.J.J.& Punt, P.J. (1991) " Gene transfer systems and vectordevelopment for filamentous fungi; in:Applied Molecular Genetics of Fungi, Peberdy, J.F.et al.; eds.; p.1-28, Cambridge University Press:Cambridge) algae or metaphyte cell be (referring to Schmidt, R.and Willmitze R, L. (1998) High efficiencyAgrobacterium tumefaciens-mediated transformation of Arabidopsisthaliana leaf and cotyledon explants " Plant Cell Rep.:583-586) or mammalian cell.Host cell appropriate is discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, CA (1990).In addition, recombinant expression carrier can transcription and translation in vitro, such as T7 promoter is used to adjust sequence and T7 polymerase.

Protein expression in prokaryotic cell often instructs the expression of fused protein or non pregnant women matter by these promoters that the carrier containing composing type perhaps inducible promoter carries out.Fusion vector adds the amino acid of certain amount in coding protein, usually in the amino terminal of recombinant protein.This fusion vector has 3 typical uses: 1) increasing the expression of recombinant protein；2) increase the dissolubility of recombinant protein；Aglucon with affinity purification 3) is used as, helps fusion protein purification.In fusion expression vector, protein cleavage site is often introduced into the junction of fusion part and recombinant protein, so that after being purified into fused protein, recombinant protein can be partially separated open with merging.This enzyme and their cognate recognition sequence, including Xa factor, fibrin ferment and enterokinase.

Typical fusion expression vector includes pGEX (Pharmacia Biotech Inc；Smith, D.B.and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,) and pRIT5 (Pharmacia MA, Piscataway, NJ), they respectively with target recombinant protein Glutathione S-transferase (GST), maltose E binding protein or a-protein.In one embodiment, pts protein coded sequence is cloned into pGEX expression vector, generates the carrier of an encoding fusion protein, which includes GST- thrombin cleavage site-X protein matter from the end N- to the end C-.Glutathione-agarose resin can be used in fusion protein, passes through affinitive layer purification.The recombination pts protein separated with GST, can be by being obtained with fibrin ferment splitting fused protein.

The example of suitable Escherichia coli inducible non-fusion expression vector includes pTrc (Amann etal., (1988) Gene 69:301-315), pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pSH1, pSH2, pPLc236, pMBL24, pLG200, pUR290, pIN-III 113-B1, λ gt11, pBdC1, with pET 11d (Studier et al., Gene ExpressionTechnology:Methods in Enzymology 185, Acad Emic Press, San Diego, California (1990) 60-89；And Pouwels et al., eds. (1985) Cloning Vectors.Elsevier:New York IBSN 0 444 904018).The target gene of pTrc carrier is expressed, the transcription of the host RNA polymerase dependent on hybrid trp-lac fusion promoter.The target gene of pET 11d carrier is expressed, the transcription for the T7gn10-lac promoter, fusion that the viral rna polymerase (T7gn1) dependent on coexpression mediates.The varial polymerases are provided by the λ bacteriophage being resident in host strain BL21 (DE3) or HMS174 (DE3), which contains the T7gn1 gene under the control of 5 promoter transcription of lacUV.Conversion for other bacterial species can choose suitable carrier.For example, as it is known that plasmid pIJ101, pIJ364, pIJ702 and pIJ361 conversion streptomycete are effective, and plasmid pUB110, pC194 or pBD214 are suitble to convert rod-shaped strain.Several plasmids for helping hereditary information to be transferred to corynebacteria include pHM1519, pBL1, (Pouwels et al., eds. (1985) Cloning Vectors, Elsevier:NewYork IBSN 0 444 is 904018) by pSA77 or pAJ667.

It is a kind of to greatest extent increase recombinant protein expression scheme be, such protein is expressed in host cell, the protein has the ability (Gottesman for the proteolytic cleavage recombinant protein that will not weaken, S., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).Another scheme is to change the nucleic acid sequence of insertion expression vector amplifying nucleic acid, so that the codon of each amino acid is that the selected bacterium for expression preferentially uses, such as Corynebacterium glutamicum (Wada et al. (1992) Nucleic Acids Res.20:2111-2118).This change of nucleic acid sequence of the present invention can be carried out by standard DNA synthesis technique.

In another embodiment, pts protein expression vector is Yeast expression carrier.The example of carrier of the yeast S.cerivisae for expression include, pYepSec1 (Baldari, et al., (1987) Embo is J.6:229-234), 2 μ, pAG-1, Yep6, Yep13, pEMBK Ye23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), with pYES2 (Invitrogen Corporation, San Diego, CA).It is suitble in other fungies for constructing, such as in filamentous fungi, the carrier and method of the carrier used, it is specified in following documents including those: van den Hondel, C.A.M.J.J.&Punt, P.J. (1991) " Gene transfer systems and vector development forfilamentous fungi; in:Applied Molecular Genetics of Fungi; J.F.Peberdy; etal.; eds., p.1-28, Cambridge University Press:Cambr Idge, and Pouwels et al., eds. (1985) Cloning Vectors, Elsevier:New York IBSN 0 444 is 904018).

It is expressed in insect cell in addition, rhabdovirus expression vector can be used in pts protein of the present invention.In the insect cell (such as Sf9 cell) of culture, baculovirus vector for expressing protein includes that pAC series (Smith et al. (1983) Mol.Cell Biol.3:2156-2165) and pVL are serial (Lucklow and Summer (1989) Virology 170:31-39).

In another embodiment, pts protein of the present invention can express in unicellular plant cells (such as algae), or express in the plant cell of higher plant (such as seed plant, seem crop plants).The example of plant expression vector includes that those are specified in following documents: Becker, D., Kemper, E., Schell, J.and Masterson, R. (1992) " New plant binaryvectors with selectable markers located proximal to the left border ", Plant Mol.Biol.20:1195-1197；And Bevan, M.W. (1984) " Binary Agrobacterium vectors for planttransformation ", Nucl.Acid.Res.12:8711-8721, including pLGV23, pGHlac+, pBIN19, pAK2004 and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors.Elsevier:New York IBSN 0 444 904018).

It is also in another embodiment, nucleic acid of the present invention is expressed in mammalian cells using mammalian expression vector.The example of mammalian expression vector includes pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO is J.6:187-195).The control function of expression vector when using in mammals at that time, is often provided by viral regulatory elements.For example, usually used promoter comes from polyoma, adenovirus 2, cytomegalovirus and simian virus 40.Other are for prokaryotic cell and all suitable expression system of eukaryocyte, referring to Sambrook, J., Fritsh, E.F., and Maniatis, T.Molecular Cloning:ALaboratory Manual.2nd, ed.Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, 16 chapters and 17 chapters of Cold Spring Harbor, NY, 1989.

In another embodiment, the mammalian expression vector of recombination can instruct the expression (being used to express nucleic acid for example, tissue specificity adjusts element) of preferred nucleic acid in particular cell types.It is technically known that tissue specificity, which adjusts element,.The example of suitable tissue-specific promoter includes but is not limited to that (liver is special for albumin promoter；Pinkert et al. (1987) Gene Dev.1:268-277), lymph specific promoter (Calame and Eaton (1988) Adv.Immunol.43:235-275), special promoter (Banerji et al. (1983) Cell 33:729-740 of special promoter (Winoto and Baltimore (1989) EMBO is J.8:729-933) and immunoglobulin of T-cell receptors；Queen and Baltimore (1983) Cell 33:741-748), the promoter of neuron-specific (such as neurofilament promoter；Byrne and Ruddle (1989) PANS86:5473-5477), the special promoter of pancreas (Edlund et al. (1985) Science 230:912-916) and the special promoter of mammary gland (such as milk whey promoter；U.S.Patent No.4,873,316 and European Application Publication No.264,166).It also include the promoter of growth adjustment, such as muroid hox promoter (Kessel and Gruss (1990) Science249:374-379) and α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

Present invention further provides the recombinant expression carrier for containing DNA molecular of the present invention, which is cloned in expression vector with antisense orientation.That is, DNA molecular can be adjusted in sequence with being connected in the following manner for operability, that is, by way of allowing to express (passing through the transcription of DNA molecular) with the RNA molecule of PTS mRNA antisense.It can choose those adjusting sequences that antisense rna molecule continuous expression is instructed in various cell types, such as viral promotors and/or enhancer, it either can choose the adjusting sequence of continuous, organizing specific or that cell type the is special antisense RNA expression of guidance, as adjusting sequence.Antisense expression vector can exist in the form of recombinant plasmid, phasmid or attenuated virus, generate under control of the wherein antisense nucleic acid in high efficiency regulatory region domain, and activity can be determined by introducing the cell type of carrier.About using antisense gene to adjust gene expression, Weintraub, H.et al., Antisense RNA as amolecular tool for genetics analysis, Review-Trends in Genetics, Vol.1 (1) 1986 may refer to.

Another aspect of the present invention is related to being introduced into the host cell of recombinant expression carrier of the present invention.Term " host cell " and " recombinant host cell " can be used alternatingly here.The term is construed as, and refers not only to the specific cells being selected, and refers to the offspring or possible offspring of these cells.Because mutation or environment influence meeting so that certain modifications occur in successful passage, these progeny cells are also contained within the scope of term used herein it is practically impossible to identical with mother cell.

Host cell can be any protokaryon or eukaryocyte.For example, pts protein can seem in bacterial cell as Corynebacterium glutamicum, in insect cell, in yeast cells or expression in mammalian cell (such as Chinese rat ovary cell (CHO) or COS cell).Other suitable host cells are well known for being familiar with for ordinary skill.The Corynebacterium glutamicum relationship microorganism that may be used as nucleic acid and protein molecule host cell of the present invention, lists in table 3.

Carrier DNA can perhaps rotaring dyeing technology introduces protokaryon or eukaryocyte by routine transformation.As used herein, being meant that for term " conversion " and " transfection " is various well known in the art, exogenous nucleic acid (such as, linear DNA or RNA (such as, the independent gene structure of linear carrier or not carrier)) or with nucleic acid existing for carrier format (such as, plasmid, bacteriophage, phasmid, phasmid, transposons or other DNA) it is transferred to the technology of host cell, including calcium phosphate or calcium chloride co-percipitation, the transfection that DEAE- dextran mediates, lipofection or fax hole.The appropriate method of conversion or transfection host cell, it can be in Sambrook, et al. (Molecular Cloning:A Laboratory Manual.2nd, ed.., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY, 1989) it and on other laboratory manuals finds.

It is known that, by the expression vector and rotaring dyeing technology used, only sub-fraction can be integrated into exogenous DNA in its own genome for stable transfection mammalian cell.In order to identify and screen these intergrants, the gene of coding selection markers (for example, to resistance of antibiotic) is usually introduced into host cell with interested gene.Preferred selection markers include the label that those can assign drug resistance, such as G418, hygromycin and amethopterin.The nucleic acid for encoding selection markers, can be introduced into host cell, or introduce host cell on individual carrier with pts protein on the same carrier.It is introduced into the cell of nucleic acid stability transfection, drug screening identification can be used (for example, the cell merged with riddled basins can survive, and other cells are then died).

In order to create homologous recombination microorganism, carrier of the preparation containing at least partly PTS gene, the gene is with missing, addition or replaces, to change, such as functional destruction, PTS gene.Preferably this is Corynebacterium glutamicum PTS gene, but it is also possible to the homologue from relationship bacterium, even from mammal, yeast or insect.In a preferred embodiment, design vector, so that endogenous PTS gene is destroyed by functionality (that is, a yard functional protein of not being on the permanent staff according to homologous recombination；Also referred to as " knock out " carrier).Furthermore it is possible to design vector, so that endogenous PTS gene is mutated or is changed according to homologous recombination, but still encoding function protein (for example, changing upstream regulatory domains, to change the expression of endogenous PTS gene).In homologous recombination vector, the PTS Gene Partial being changed is connected with extra PTS nucleic acid in its 5 ' and 3 ' end profile, and homologous recombination is occurred between the endogenous PTS gene of external source PTS gene and microorganism that carrier carries.The PTS nucleic acid of extra side connection has enough length, homologous recombination can successfully occur with endogenous gene.Typically, in carrier containing thousands of a bases side chain DNA (5 ' and 3 ' end) (referring to, such as, Thomas, K.R., and Capecchi, M.R. (1987) Cell 51:503 for a description of homologous recombinationvectors).The carrier in microorganism (such as fax hole) and cell, the PTS gene for selecting those wherein to introduce and endogenous PTS gene are introduced, it can be with homologous recombination using technology known in the art.

In another embodiment, it can produce the recombinant microorganism containing the system that is allowed a choice, which allows to adjust the expression for introducing gene.For example, comprising PTS gene be under the control of lac operon in the carrier so that PTS gene can only express in the presence of IPTG.What this regulating system was well known.

In another embodiment, the endogenous PTS gene in host cell is destroyed (for example, by known genetic method in homologous recombination or other technologies), prevent the expression of its protein product is from occurring.In another embodiment, the PTS gene of the endogenous perhaps introducing in host cell changes through one or more point mutation, missing or inversion, but still encoding function pts protein.And in another embodiment, microorganism PTS gene one or more adjustment region (such as, promoter, repressor or elicitor) it is changed (for example, by missing, shearing, inversion or point mutation), so that the expression of PTS gene is adjusted.Ordinary skill is familiar with it is to be appreciated that the host cell containing PTS gene and protein modification described in more than one, can easily be generated, these cells are also included in the present invention using method of the invention.

Host cell of the present invention, such as the protokaryon or eukaryotic host cell of culture can be used for generating (such as expression) pts protein.Therefore, invention further provides the methods for generating pts protein using host cell of the present invention.In one embodiment, this method includes cultivating host cell of the invention in suitable culture medium (wherein to introduce the recombinant expression carrier of coding pts protein, the gene of the pts protein of encoding wild type or change is either introduced in its genome), until generating pts protein.In another embodiment, this method further comprises separating pts protein from culture medium or host cell.

C. the pts protein separated

Another aspect of the present invention relates to isolated pts protein and its biologically-active moieties." separation " perhaps " purifying " albumen or its biologically-active moiety, there is no cellular material when being produced using recombinant DNA technology, there is no precursor or other chemical substances when chemical synthesis.Term " substantially free of cellular material " includes such pts protein preparation, and wherein protein is isolated from the cellular component for the cell that natural or recombination generates the protein.In one embodiment, term " substantially free of cellular material " includes the pts protein that preparation contains at least about 30% (dry weight) non-pts protein (" contaminating protein matter " here also called), the preferred non-pts protein containing less than about 20%, even more preferably containing the non-pts protein less than about 10%, the most preferred non-pts protein containing less than about 5%.When pts protein or its biologically-active moiety are generated through recombination, preferably substantially free of culture medium, i.e. culture medium is most preferably less than about 5% less than preparing about the 20%, preferably less than 10% of protein volume.Term " substantially free of precursor perhaps other chemical substances " include such pts protein preparation wherein protein by from participating in precursor that protein synthesizes or isolated in other chemical substances.In one embodiment, term " substantially free of precursor or other chemical substances " includes pts protein of the preparation containing at least about 30% (dry weight) precursor or non-PTS chemical substance, the preferred precursor containing less than about 20% or non-PTS chemical substance, even more preferably containing the precursor less than about 10%, perhaps non-PTS chemical substance most preferably contains precursor or non-PTS chemical substance less than about 5%.In a preferred embodiment, isolated protein or its biologically-active moiety, without containing the contaminating protein matter from the same organism for obtaining pts protein.This protein is typically generated by recombinant expression, such as the recombinant expression of the Corynebacterium glutamicum pts protein in the such microorganism of Corynebacterium glutamicum.

The pts protein of the invention or its biologically-active moiety of separation, can participate in handle seems that high-energy carbon-containing molecules as glucose are transported into Corynebacterium glutamicum, perhaps the activity for participating in the Cellular Signaling Transduction Mediated in the microorganism or being listed in Table 1 with one or more.In a preferred embodiment, such amino acid sequence is contained in protein or its part, the sequence and amino acid sequence of the invention (such as, sequence table even order number a: sequence in sequence) there is sufficient homology, so that the protein or its biologically-active moiety, it has the ability to participate in handle to seem that high-energy carbon-containing molecules as glucose are transported into Corynebacterium glutamicum, or participate in the Cellular Signaling Transduction Mediated in the microorganism.The part of protein preferably refers to biologically-active moiety described herein.In another preferred embodiment, pts protein of the invention has in sequence table with even order number: the amino acid sequence listed.In another preferred embodiment, pts protein has by nucleotide sequence coded amino acid sequence, the nucleotide sequence hybridizes with nucleotide sequence (for example, sequence table odd serial numbers: a sequence in sequence) of the invention, such as hybridizes under strict conditions.In another preferred embodiment, pts protein has by such nucleotide sequence coded amino acid sequence, the nucleotide sequence and a nucleic acid sequence of the invention or its part, have at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% homology, preferably have at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% homology, more preferably have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89 % perhaps 90% or 91%, 92%, 93%, 94%, and even more preferably have at least about 95%, 96%, 97%, 98%, 99% or higher homology.Range or consistency value (for example, consistency of the consistency of 70-90% or 80-95%) between above-cited value, also intentional includes in the present invention.For example, intentional contains such consistency value range, these ranges are the combinations of the above-cited upper limit and/or lower limit value.Currently preferred pts protein it is also preferred that there is at least one PTS activity described herein.Such as, a kind of currently preferred pts protein includes such nucleotide sequence coded amino acid sequence, the nucleotide sequence and nucleotide sequence hybridization of the invention, such as hybridize under strict conditions, and it seems that high-energy carbon-containing molecules as glucose are transported into Corynebacterium glutamicum that the sequence, which can participate in handle, perhaps the activity for participating in the Cellular Signaling Transduction Mediated in the microorganism or being listed in Table 1 with one or more.

In other embodiments, pts protein and amino acid sequence of the invention (such as, sequence table even order number a: sequence in sequence) there is sufficient homology, and the functional activity with amino acid sequence protein of the present invention, as as being described in detail the above part I, amino acid sequence is different due to naturally change or being mutated.Therefore, in another embodiment, pts protein is such protein, the amino acid sequence and all aminoacid sequence of the invention that it has, have at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% homology, preferably have at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% homology, more preferably have at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90%, or 91%, 92%, 93%, 94%, and even more preferably have at least about 95%, 96%, 97%, 98%, 99% or higher homology, and there is at least one PTS activity described herein.Range or consistency value (for example, consistency of the consistency of 70-90% or 80-95%) between above-cited value, also intentional includes in the present invention.For example, intentional contains such consistency value range, these ranges are the combinations of the above-cited upper limit and/or lower limit value.In another embodiment, the present invention is related with such overall length Corynebacterium glutamicum protein, and the protein and amino acid sequence of the invention have sufficient homology.

The biologically-active moiety of pts protein includes such polypeptide, the polypeptide contains the amino acid sequence from pts protein amino acid sequence, such as, sequence table even order number: amino acid sequence or amino acid sequence with pts protein homologous protein, amino acid more less than overall length pts protein or overall length pts protein homologous protein is contained in the part, and shows at least one pts protein activity.Typical biologically-active moiety (peptide, for example, amino acid length be seem 5,10,15,20,30,35,36,37,38,39,40,50,100 or more peptides) include one there is the active structural domain of at least one pts protein or primitive.In addition, other biological activities part, wherein the other parts of protein have been deleted, and can be prepared by recombinant technique, and identify its one or more kinds of activity described herein.The biologically-active moiety of preferred pts protein, the biologically active structural domain/primitive selected containing one or more or its part.

Pts protein is produced preferably through recombinant DNA technology.For example, expression vector is introduced into host cell (as described above) and expresses pts protein in host cell by the cloned nucleic acid molecule of coding protein into expression vector (as described above).Then pts protein is separated from cell using standard protein purification technique according to suitable purification schemes.In addition to recombinant expression, standard peptide synthesis methods chemical synthesis pts protein, polypeptide or peptide can be used.In addition, natural pts protein can be separated from cell (such as endothelial cell), such as using anti-PTS antibody, which can be used pts protein or its part of the invention and passes through standard technique generation.

Present invention provides PTS chimeric protein or fusion proteins.As used herein, PTS " chimeric protein " or " fusion protein " contain the PTS polypeptide being operatively connected on non-PTS polypeptide." PTS polypeptide " refers to the polypeptide containing PTS related amino acid sequence, and " non-pts protein " refers to the polypeptide containing such protein related amino acid sequence, the protein and the not basic homology of pts protein, for example, the protein different from pts protein from same or different organism.In fused protein, term " operability connection " is meant that pts protein is in-frame merge between each other with non-pts protein.Non- PTS polypeptide can be fused to the end N- or the end C- of PTS polypeptide.For example, in one embodiment, fused protein is DST-PTS fusion protein, wherein PTS sequence is fused to the end C- of GST sequence.The fused protein helps to recombinate the purifying of pts protein.In another embodiment, fused protein is the pts protein for having Heterologous signal sequences in its end N-.In certain host cells (such as mammalian host cell), the expression and/or secretion of pts protein can be increased by using Heterologous signal sequences.

It is preferred that chimeric protein or fusion protein of the invention is generated by standard recombinant dna technology.Such as, it is linked together according to the DNA fragmentation that routine techniques encodes different polypeptide sequences by in-frame, such as, it is connected using the end of blunt end or staggered end, it is digested using restriction enzyme to provide suitable end, use cohesive end filling-in as suitable end, using alkaline phosphatase treatment to avoid undesirable connection, and is connected using enzymatic.In another embodiment, it includes automatic dna synthesizer synthesis fusion that routine techniques, which can be used,.In addition, the PCR amplification that anchor primer carries out genetic fragment can be used, anchor primer can increase the complementary jag between two consecutive gene fragments, consecutive gene can then carry out annealing and expand again and generate chimeric gene sequence (referring to, such as, Current Protocols in MolecularBiology, eds.Ausubel et al.John Wiley&Sons:1992).In addition, the expression vector of many encoded fusion parts (such as gst polypeptide) is that business provides.PTS- code nucleic acid can be cloned into this expression vector, so that merging, part is in-frame to be connected on pts protein.

The homologue of pts protein can be generated by mutation, such as the discontinuity point mutation or shearing of pts protein.As used herein, term " homologue " refers to the variant form of pts protein, they may be used as the active agonist of pts protein or antagonist.The bioactivity that the agonist of pts protein substantially can have pts protein identical or partial.The antagonist of pts protein can inhibit the one or more activity of the naturally occurring form of pts protein, for example, passing through downstream with the PTS system comprising pts protein or upstream member's competitive binding.Therefore, Corynebacterium glutamicum pts protein and its homologue of the invention, the activity of adjustable one or more of carbohydrate transporting pathway, or adjust the activity for the Cellular Signaling Transduction Mediated approach that pts protein plays a role in the microorganism.

In a further embodiment, the homologue of pts protein can pass through the combinatorial libraries of screening pts protein mutant, such as shearing mutant, Lai Jianding pts protein agonist or antagonist activities.In one embodiment, the diverse libraries of PTS variant generate and associativity is mutated in nucleic acid level, and are encoded by diversity gene library.The diverse libraries of PTS variant can pass through, such as, the oligonucleotide cocktail enzymatic of synthesis is connected in gene order, so that the degeneracy set of potential PTS sequence is as single polypeptide, or the wherein set of the bigger fused protein (such as phage display) containing PTS arrangement set, it can express.There are various methods to can be used for generating potential PTS homologue library from degeneracy oligonucleotide sequences.The chemical synthesis of degeneracy gene order can be carried out with automatic dna synthesizer, then synthesized gene and be connected in suitable expression vector.The use of gene degeneracy set, the full sequence of potential PTS arrangement set needed for the offer for allowing to mix encodes.The method for synthesizing degeneracy oligonucleotide is technically known (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3；Itakura et al. (1984) Annu.Rev.Biochem.53:323；Itakura et al. (1984) Science 198:1056；Ike et al. (1983) Nucleic Acid Res.11:477).

In addition, the library of coding pts protein segment, can be used for generating the diverse populations of PTS segment, which is used to screen and select the homologue of pts protein.In one embodiment, the library of coding sequence fragment can generate in this way, i.e. under conditions of about per molecule only generates a notch, with the double stranded PCR fragment of nucleic acid enzymatic treatment PTS coded sequence, denatured double stranded dna, for renaturation DNA to form double-stranded DNA, which may include the sense/antisense pair that the product for having notch from difference is formed, single stranded portion is removed by S1 nucleic acid enzymatic treatment in the double helix re-formed, and the frag-ment libraries finally obtained are connected in expression vector.In this way, the expression library of the available coding end N-, the end C- and different size pts protein intermediate segment.

The technology with institute's picking up characteristics gene product in many technologies of the gene product in the combinatorial libraries obtained by point mutation or shearing, and screening cDNA library is screened, is all technically known.These technologies are suitable for the quick screening of the gene library obtained by PTS homologue combinatorial mutagenesis.Screen the most widely used technology of large-scale gene pool, it can be used in high throughput analysis, including genomic libraries of clones into reproducible expression vector, with obtained vector library conversion carrier library, and combination gene is expressed under certain condition, required active detection helps to encode the separation of the carrier of detected product gene under this condition.Assemblage mutation (REM) is returned, a kind of new technology increasing function mutation body frequency in library can be with screening analysis together for identifying PTS homologue (Arkin and Yourvan (1992) PANS 89:7811-7815；Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

In another embodiment, using method known in the art, the analysis based on cell can be used for analyzing the library diversity PTS.

D. application and method of the invention

Nucleic acid molecules, protein, protein homology object, fused protein, primer, carrier and host cell described herein can be applied in following one or more kinds of methods: identification Corynebacterium glutamicum and relationship microorganism；Draw the Genome Atlas of Corynebacterium glutamicum relationship organism；The sequence interested of identification and positioning Corynebacterium glutamicum；Study on Evolution；Determine the required region of the function of pts protein；Pts protein Active Regulation；PTS pathway activities are adjusted；Required compound, for example, fine chemical cell production adjusting.

PTS nucleic acid molecules of the present invention have various uses.Firstly, they can be used for identifying whether a kind of organism is Corynebacterium glutamicum or its close relative organism.They can be used for identification mixed microorganism group Glutamic Acid bar bacterium or the presence of its relationship organism.The present invention provides the nucleic acid sequences of many Corynebacterium glutamicum genes；Under strict conditions, using across the probe to Corynebacterium glutamicum specific gene, the genomic DNA extracted from single or mixed microorganism culture is detected, can determine that the organism whether there is.

Although Corynebacterium glutamicum itself is non-pathogenic, it is related to pathogenic type, such as corynebacterium diphtheriae.Corynebacterium diphtheriae is the etiology of diphtheria, and diphtheria is a kind of infection quickly grow, is acute, having a fever, it is related to local symptom and system symptom.When obtaining this disease, local patholoic change occurs for the upper respiratory tract, and including epithelial cell necrotic injury；Bacteria secrete toxins, toxin spread to the distal tissues of body susceptible from lesion.These tissue include heart, muscle, peripheral nerve, adrenal gland, kidney, liver and spleen, wherein as protein synthesis be suppressed and caused by go bad sexually revise, will lead to the system symptom of the disease.Diphtheria keeps high incidence in many areas in the world, these areas include the independent state in Africa, Asia, Eastern Europe and the former Soviet Union.From nineteen ninety, in the continued popularity of latter two regional diphtheria, at least 5 are resulted in, 000 people is dead.

In one embodiment, the present invention exists with corynebacterium diphtheriae in identification subject or active method is related.This method includes identifying in subject one or more of nucleic acid of the invention or amino acid sequence (for example, the odd number being listed in sequence table respectively perhaps even order sequence) to detect the presence or activity of corynebacterium diphtheriae in subject.Corynebacterium glutamicum and corynebacterium diphtheriae are akin bacteriums, and many nucleic acid and protein molecule in Corynebacterium glutamicum are the homologue of corynebacterium diphtheriae amplifying nucleic acid and protein molecule, therefore can be used for the corynebacterium diphtheriae in detection subject.

Nucleic acid and protein molecule of the invention is also used as the label of specific genome area.This is not only useful when drawing Genome Atlas, but also can be used for the research of Corynebacterium glutamicum protein function.For example, Corynebacterium glutamicum gene group can be digested in order to identify specific Corynebacterium glutamicum DNA binding protein genome area in combination, segment and DNA binding protein are incubated for.Segment in conjunction with protein can be further with nucleic acid molecules detection of the invention, it is preferable to use easily detection marks；The combination of these nucleic acid molecules and genomic fragment can position position of the segment on Corynebacterium glutamicum gene group map, moreover, helping quickly to determine protein nucleic acid sequence in combination when carrying out multi-pass operation using different enzymes.In addition, nucleic acid molecules of the present invention can have sufficient homology with relationship type, so that these nucleic acid molecules can be used as the label of building relationship bacterial genomes map, such as brevibacterium.

PTS nucleic acid molecules of the invention can be used for evolving again and protein structure research.The carbohydrate capturing system that molecule of the present invention participates in, is used by various bacteriums；The sequence of nucleic acid molecules of similar enzyme is encoded in other organisms with those by comparing sequence of nucleic acid molecules of the present invention, can be evaluated whether the evolution correlation of organism.Similar, this comparison allows to estimate conserved sequence region and non-conserved sequences region, this can contribute to determine in protein for region necessary to enzyme function.Such determination is valuable for protein engineering research, and can indicate that those protein can endure mutation without losing function.

The operation of PTS nucleic acid molecules of the present invention can cause with the generation with the pts protein of wild type pts protein different function.The efficiency or activity that these protein can be improved can be allowed to either reduce its efficiency or activity to appear in cell than usual more numbers.

The present invention provides the methods for screening the adjustable active molecule of pts protein, these molecules or by with protein itself or substrate interaction, perhaps active to adjust pts protein in conjunction with the gametophyte of pts protein or through the transcription or translation of adjusting PTS nucleic acid molecules of the present invention.In the method, the microorganism for expressing one or more kinds of pts proteins, contacts with one or more kinds of test compounds, and assesses effect of the every kind of test compound for pts protein activity or expression.

PTS molecule of the present invention can be modified, so that yield, production and/or the production efficiency of one or more kinds of chemical substances are improved.For example, optimize its activity by the pts protein that modification participates in glucose uptake, it can increase glucose uptake amount or glucose is transported rate into cell.The degradation of glucose inside cells and other carbohydrates provides the biochemical reaction that energy pushes energy unfavorable, such as those are related to the reaction of fine chemical biosynthesis.Degradation also provides intermediate or precursor molecule necessary to the certain fine chemicals of biosynthesis, such as amino acid, vitamin and co-factor.By modifying PTS molecule of the present invention to increase the quantity of intracellular high energy carbon molecules, the energy of metabolic pathway is executed necessary to producing one or more kinds of fine chemicals so as to not only increase, but also endocellular metabolism object library required for this production can be increased.Opposite, the catabolite of some carbohydrates contains a kind of compound, the compound is served only for such metabolic pathway, the approach is vied each other because of enzyme, co-factor or intermediate with the approach for generating required fine chemical is used as, it, can be with the negative regulator approach by reducing the input of these carbohydrates.

In addition, PTS molecule of the invention can participate in one or more kinds of Cellular Signaling Transduction Mediated approach, these approach can influence the yield and/or production efficiency of one or more kinds of fine chemicals in Corynebacterium glutamicum.Such as, once there is sufficient amount of carbohydrate into the cell, from inputted in extracellular medium protein necessary to one or more kinds of carbohydrates (such as, HPr, Enzyme I, or one of Enzyme II complex ingredient) be often translated after modify, to prevent them from sugar is input into the cell again.Such a example appears in Escherichia coli, and the high level of intracellular fructose 1,6-diphosphate leads to the phosphorylation of HPr serine -46, prevents the molecule from participating in the transhipment of any carbohydrate again.However, horizontal in this intracellular carbohydrate that movement system is closed, for maintain the normal function of cell be it is enough, this may limit the excessive production of required fine chemical.Therefore, it is desirable for modifying pts protein of the invention in this way, i.e. so that they are no longer effective to this negative regulator, to allow can achieve the higher intracellular concentration of one or more kinds of carbohydrates, and, extending allows to obtain one or more kinds of fine chemicals from the organism containing this mutation pts protein more effectively to produce or higher yield.

The above-mentioned list for leading to the increased PTS mutation scheme of required compound production, is not meant to be limited only to this；The variation of these mutation schemes is known better for person skilled in the art.By these mechanism, nucleic acid of the invention and protein molecule can be used for generating Corynebacterium glutamicum or its relationship bacterial strain of expression mutation PTS nucleic acid and protein molecule, to increase yield, production and/or the production efficiency of required compound.The required compound can be any natural products of Corynebacterium glutamicum, this includes the final product of biosynthesis pathway and the intermediate of naturally occurring metabolic pathway, and is not molecule that is naturally occurring in Corynebacterium glutamicum metabolism but being generated by Corynebacterium glutamicum strain of the present invention.

The present invention is further illustrated by following instance, these examples are not construed as being limited only to this.Content in the application in cited all bibliography, patent application, patent, the patent application delivered, table and sequence list is all merged into bibliography hereby.

Table 1: gene by the invention

Phosphoenolpyruvate: sugar phosphotransferase system

Nucleotide SEQ ID NO	Amino acid SEQ ID NO	Number	Contig	NT starting	NT is terminated	Function
Nucleotide SEQ ID NO	Amino acid SEQ ID NO	Number	Contig	NT starting	NT is terminated	Function	1	2	RXS00315				PTS system, sucrose-specificity IIABC ingredient (EIIABC-SCR) (sucrose-permease IIABC ingredient (phosphotransferase II, ABC ingredient) (EC 2.7.1.69)
3	4	F RXA00315	GR00053	6537	5452	PTS system, BETA- glucosides-specificity IIABC ingredient (EIIABC-BGL) (BETA- glucosides-permease IIABC ingredient) (phosphotransferase II, ABC ingredient) (EC 2.7.1.69)	1	2	RXS00315
3	4	F RXA00315	GR00053	6537	5452		5	6	RXA01503	GR00424	10392	10640	PTS system, BETA- glucosides-specificity IIABC ingredient (EIIABC-BGL) (BETA- glucosides-permease IIABC ingredient) (phosphotransferase II, ABC ingredient) (EC 2.7.1.69)
7	8	RXN01299	VV0068	11954	9891	PTS system, fructose-specificity IIBC ingredient (EC 2.7.1.69)	5	6	RXA01503	GR00424	10392	10640
7	8	RXN01299	VV0068	11954	9891		9	10	F RXA01299	GR00375	6	446	PTS system, fructose-specificity IIBC ingredient (EC 2.7.1.69)
11	12	F RXA01883	GR00538	2154	2633	PTS system, fructose-specificity IIBC ingredient (EC 2.7.1.69)	9	10	F RXA01299	GR00375	6	446
11	12	F RXA01883	GR00538	2154	2633		13	14	F RXA01889	GR00540	77	631	PTS system, fructose-specificity IIBC ingredient (EC 2.7.1.69)
15	16	RXA00951	GR00261	564	172	PTS system, mannitol (secret)-specificity IIA ingredient (EIIA- (C) MTL) (mannitol (secret)-permease IIA ingredient) (phosphotransferase II, A ingredient) (EC 2.7.1.69)	13	14	F RXA01889	GR00540	77	631
15	16	RXA00951	GR00261	564	172		17	18	RXN01244	VV0068	14141	15844	Phosphoenolpyruvate-protein phosphotransferase (EC 2.7.3.9)
19	20	F RXA01244	GR00359	4837	3329	Phosphoenolpyruvate-protein phosphotransferase (EC 2.7.3.9)	17	18	RXN01244	VV0068	14141	15844	Phosphoenolpyruvate-protein phosphotransferase (EC 2.7.3.9)
19	20	F RXA01244	GR00359	4837	3329	Phosphoenolpyruvate-protein phosphotransferase (EC 2.7.3.9)	21	22	RXA01300	GR00375	637	903	Phosphocarrier protein HPR
23	24	RXN03002	VV0236	1437	1844	PTS system, mannitol (secret)-specificity IIA ingredient (EIIA- (C) MTL) (mannitol (secret)-permease IIA ingredient) (phosphotransferase II, A ingredient) (EC 2.7.1.69)	21	22	RXA01300	GR00375	637	903	Phosphocarrier protein HPR
23	24	RXN03002	VV0236	1437	1844		25	26	RXC00953	VV0260	1834	1082	Transmembrane protein in PTS system
27	28	RXC03001				Transmembrane protein in PTS system	25	26	RXC00953	VV0260	1834	1082	Transmembrane protein in PTS system
27	28	RXC03001				Transmembrane protein in PTS system	29	30	RXN01943	VV0120	4326	6374	PTS system, glucose-specificity IIABC ingredient (EC 2.7.1.69)
31	32	F RXA02191	GR00642	3395	4633	Phosphoenolpyruvate carbohydrate phosphotransferase	29	30	RXN01943	VV0120	4326	6374
31	32	F RXA02191	GR00642	3395	4633	Phosphoenolpyruvate carbohydrate phosphotransferase	33	34	F RXA01943	GR00557	3944	3540	Crr gene；Phosphotransferase system glucose-enzyme-specific III

The gene of table 2:GENBANK identification

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	A09073	ppg	Phosphoric acid enol pyruvic acid carboxylase	Bachmann, B.et al. " DNA fragment coding for phosphoenolpyruvat corboxylase; recombinant DNA carrying said fragment; strains carrying the recombinant DNA and method for producing L-aminino acids using said strains, " Patent:EP 0358940-A 303/21/90
A45579, A45581, A45583, A45585 A45587		Threonine dehydratase	Moeckel, B.et al. " Production of L-isoleucine by means of recombinant micro-organisms with deregulated threonine dehydratase, " Patent:WO 9519442-A 507/20/95	A09073	ppg	Phosphoric acid enol pyruvic acid carboxylase
A45579, A45581, A45583, A45585 A45587		Threonine dehydratase		AB003132	murC；ftsQ；ftsZ		Kobayashi, M.et al. " Cloning, sequencing, and characterization of the ftsZ gene from coryneform bacteria; " Biochem.Biophys.Res.Commun., 236 (2): 383-388 (1997)
AB015023	murC；ftsQ		Wachi, M.et al. " A murC gene from Coryneform bacteria, " Appl.Microbiol. Biotechnol., 51 (2): 223-228 (1999)	AB003132	murC；ftsQ；ftsZ
AB015023	murC；ftsQ			AB018530	dtsR		Kimura, E.et al. " Molecular cloning of a novel gene; dtsR; which rescues the detergent sensitivity of a mutant derived from Brevibacterium lactofermentum; " Biosci.Biotechnol.Biochem., 60 (10): 1565-1570 (1996)
AB018531	dtsR1；dtsR2			AB018530	dtsR
AB018531	dtsR1；dtsR2			AB020624	murI	D-Glu racemase
AB023377	tkt	Transketolase		AB020624	murI	D-Glu racemase
AB023377	tkt	Transketolase		AB024708	gltB；gltD	Glutamine 2-oxoglutaric acid transaminase is big and small subunit
AB025424	acn	Aconitase		AB024708	gltB；gltD
AB025424	acn	Aconitase		AB027714	rep	Replication protein
AB027715	rep；aad	Replication protein；Aminoglycoside adenyl transferase		AB027714	rep	Replication protein
AB027715	rep；aad	Replication protein；Aminoglycoside adenyl transferase		AF005242	argC	N- acetyl aspartate -5- semialdehyde dehydrogenase
AF005635	glnA	Glutamine synthelase		AF005242	argC	N- acetyl aspartate -5- semialdehyde dehydrogenase
AF005635	glnA	Glutamine synthelase		AF030405	hisF	Cyclase
AF030520	argG	Argininosuccinate synthetase		AF030405	hisF	Cyclase
AF030520	argG	Argininosuccinate synthetase		AF031518	argF	Ornithine transcarbamylase
AF036932	aroD	3- dehydroquinate dehydratase		AF031518	argF	Ornithine transcarbamylase

GenBank^TM

Gene Name

Gene function

Bibliography

Searching number
Searching number				AF038548	pyc	Pyruvate carboxylase
AF038651	dciAE；apt；rel	Two peptide-binding proteins；Adenine phosphoribosyl transferase；GTP pyrophosphokinase	Wehmeier, L.et al. " The role of the Corvnebacterium glutamicum rel gene in (p) ppGpp metabolism, " Microbiology, 144:1853-1862 (1998)	AF038548	pyc	Pyruvate carboxylase
AF038651	dciAE；apt；rel			AF041436	argR	Arginine repressor
AF045998	impA	Inositol monophosphate phosphorylase		AF041436	argR	Arginine repressor
AF045998	impA	Inositol monophosphate phosphorylase		AF048764	argH	Argininosuccinate lyase
AF049897	argC；argJ；argB； argD；argF；argR； argG；argH	N- acetylglutamyl reductase；Ornithine acetyltransferase；N-acetylglutamat kinases；Acetylornithice transminase；Ornithine transcarbamylase；Arginine repressor；Argininosuccinate synthase；Argininosuccinate lyase		AF048764	argH	Argininosuccinate lyase
AF049897	argC；argJ；argB； argD；argF；argR； argG；argH			AF050109	inhA	Alkene acyl-acyl carrier protein reductase
AF050166	hisG	ATP phosphoribosyltransferase		AF050109	inhA	Alkene acyl-acyl carrier protein reductase
AF050166	hisG	ATP phosphoribosyltransferase		AF051846	hisA	Phosphoribosyl formimino group -5- amino-ribose 1-phosphate -4- imidazoles carbamyl isomerase
AF052652	metA	Homoserine O- transacetylase	Park, S.et al. " Isolation and analysis of metA; a methionine biosynthetic gene encoding homoserine acetyltransferase in Corynebacterium glutamicum; " Mol. Cells., 8 (3): 286-294 (1998)	AF051846	hisA
AF052652	metA	Homoserine O- transacetylase		AF053071	aroB	Dehydroquinate synthase
AF060558	hisH	Glutamine amide transferase		AF053071	aroB	Dehydroquinate synthase
AF060558	hisH	Glutamine amide transferase		AF086704	hisE	Phosphoribosyl-ATP- pyrophosphohydrolase
AF114233	aroA	5- enolpyruvylshikimate 3- phosphate synthase		AF086704	hisE	Phosphoribosyl-ATP- pyrophosphohydrolase
AF114233	aroA	5- enolpyruvylshikimate 3- phosphate synthase		AF116184	panD	L-Aspartic acid-alpha- decarboxylation enzyme precursor	Dusch, N.et al. " Expression of the Corynebacterium glutamicum panD gene encoding L-aspartate-alpha-decarboxylase leads to pantothenate overproduction in Escherichia coli; " Appl.Environ.Microbiol., 65 (4) 1530- 1539 (1999)

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	AF124518	aroD；aroE	3-dehydroquinase；Shikimate dehydrogenase
AF124600	aroC；aroK；aroB；	Chorismate synthase；Shikimate kinase；3- dehydroquinic acid		AF124518	aroD；aroE	3-dehydroquinase；Shikimate dehydrogenase

	pepQ	Synthase；The cytosolic peptidase of supposition
	pepQ	Synthase；The cytosolic peptidase of supposition		AF145897	inhA
AF145898	inhA			AF145897	inhA
AF145898	inhA			AJ001436	ectP	Ectoine, glycinebetaine, proline transport	Peter, H.et al. " Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes:Identification; sequencing; and characterization of the proline/ectoine uptake system; ProP; and the ectoine/proline/glycine betaine carrier, EctP, " J. Bacteriol., 180 (22): 6005-6012 (1998)
AJ004934	dapD	Tetrahydro 2, dipicolimic acid 2 succinyl enzyme (imperfect i)	Wehrmann, A.et al. " Different modes of diaminopimelate synthesis and their role in cell wall integrity:A study with Corynebacterium glutamicum; " J. Bacteriol., 180 (12): 3159-3165 (1998)	AJ001436	ectP	Ectoine, glycinebetaine, proline transport
AJ004934	dapD			AJ007732	ppc；secG；amt；ocd； soxA	Phosphoenolpyruvate-carboxylase；；High-affinity ammonia absorbs albumen；The ornithine of supposition-ring decarboxylase；Sarcosine oxidase
AJ010319	FtsY, glnB, glnD；srp； amtP	Participate in cell division；PII albumen；Uridyltransferase (uridine acyl-elimination enzyme)；Signal recognition particle；Low-affinity ammonia absorbs albumen	Jakoby, M.et al. " Nitrogen regulation in Corynebacterium glutamicum；Isolation of genes involved in biochemical characterization of corresponding proteins, " FEMS Microbiol., 173 (2): 303-310 (1999)	AJ007732	ppc；secG；amt；ocd； soxA
AJ010319	FtsY, glnB, glnD；srp； amtP			AJ132968	cat	Chloramphenicol acetyltransferase
AJ224946	mqo	L MALIC ACID: quinone oxidoreductase	Molenaar, D.et al. " Biochemical and genetic characterization of the membrane-associated malate dehydrogenase (acceptor) from Corynebacteriun glutamicum; " Eur.J.Biochem., 254 (2): 395-403 (1998)	AJ132968	cat	Chloramphenicol acetyltransferase
AJ224946	mqo	L MALIC ACID: quinone oxidoreductase		AJ238250	ndh	Nadh dehydrogenase
AJ238703	porA	Porin	Lichtinger, T.et al. " Biochemical and biophysical characterization of the cell wall porin of Corynebacterium glutamicum:The channel is formed by a low molecular mass polypeptide; " Biochemistry, 37 (43): 15024-15032 (1998)	AJ238250	ndh	Nadh dehydrogenase
AJ238703	porA	Porin		D17429		Transposable element IS31831	Vertes, A.A.et al. " Isolation and characterization of IS31831; a transposable element from Corynebacterium glutamicum, " Mol.Microbiol., 11 (4): 739-746 (1994)

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	D84102	odhA	OdhA	Usuda, Y.et al. " Molecular cloning of the Corynebacterium glutamicum (Brevibacterium lactofermentum AJ12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase; " Microbiology, 142:3347-3354 (1996)
E01358	hdh；hk	Homoserine dehydrogenase；Homoserine kinase	Katsumata, R.et al. " Production of L-thereonine and L-isoleucine, " Patent:JP 1987232392-A 1 10/12/87	D84102	odhA	OdhA

E01359		Homoserine kinase gene upstream from start codon	Katsumata, R.et al. " Production of L-thereonine and L-isoleucine, " Patent:JP 1987232392-A 2 10/12/87
E01359		Homoserine kinase gene upstream from start codon		E01375	Tryptophan operon
E01376	trpL；trpE	Leader peptide；Anthranilate synthase	Matsui, K.et al. " Tryptophan operon; peptide and protein coded thereby; utilization of tryptophan operon gene expression and production of tryptophan, " Patent:JP 1987244382-A 1 10/24/87	E01375	Tryptophan operon
E01376	trpL；trpE	Leader peptide；Anthranilate synthase		E01377	Tryptophan operon promoter and operator region	Matsui, K.et al. " Tryptophan operon; peptide and protein coded thereby; utilization of tryptophan operon gene expression and production of tryptophan, " Patent:JP 1987244382-A 1 10/24/87
E03937		Biotin-synthase	Hatakeyama, K.et al. " DNA fragment containing gene capable of coding biotin synthetase and its utilization, " Patent:JP 1992278088-A 1 10/02/92	E01377	Tryptophan operon promoter and operator region
E03937		Biotin-synthase		E04040	Diaminopelargonic acid transaminase	Kohama, K.et al. " Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization, " Patent:JP 1992330284-A 1 11/18/92
E04041		Dethiobiotin synthetase	Kohama, K.et al. " Gene coding diaminopelargonic acid aminotransferase and desthiobiotin synthetase and its utilization, " Patent:JP 1992330284-A 1 11/18/92	E04040	Diaminopelargonic acid transaminase
E04041		Dethiobiotin synthetase		E04307	Flavum Aspartase	Kurusu, Y.et al. " Gene DNA coding aspartase and utilization thereof, " Patent:JP 1993030977-A 1 02/09/93
E04376		Isocitratase	Katsumata, R.et al. " Gene manifestation controlling DNA, " Patent:JP 1993056782-A 3 03/09/93	E04307	Flavum Aspartase
E04376		Isocitratase		E04377	Isocitratase N- terminal fragment	Katsumata, R.et al. " Gene manifestation controlling DNA, " Patent:JP 1993056782-A 3 03/09/93
E04484		Prephenate dehydratase	Sotouchi, N.et al. " Production of L-phenylalanine by fermentation, " Patent:JP 1993076352-A 2 03/30/93	E04377	Isocitratase N- terminal fragment
E04484		Prephenate dehydratase		E05108	Aspartokinase	Fugono, N.et al. " Gene DNA coding Aspartokinase and its use, " Patent:JP 1993184366-A 1 07/27/93
E05112		Dihydro-dipichorinate synzyme	Hatakeyama, K.et al. " Gene DNA coding dihydrodipicolinic acid synthetase and its use, " Patent:JP 1993184371-A 1 07/27/93	E05108	Aspartokinase

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	E05776	Diaminopimelate dehydrogenase	Kobayashi, M.et al. " Gene DNA coding Diaminopimelic acid dehydrogenase and its use, " Patent:JP 1993284970-A 1 11/02/93
E05779		Threonine synthase	Kohama, K.et al. " Gene DNA coding threonine synthase and its use, " Patent:JP 1993284972-A 1 11/02/93	E05776	Diaminopimelate dehydrogenase

E06110		Prephenate dehydratase	Kikuchi, T.et al. " Production of L-phenylalanine by fermentation method, " Patent:JP 1993344881-A 1 12/27/93
E06110		Prephenate dehydratase		E06111	It is mutated prephenate dehydratase	Kikuchi, T.et al. " Production of L-phenylalanine by fermentation method, " Patent:JP 1993344881-A 1 12/27/93
E06146		Acetohydroxy acid synthetase	Inui, M.et al. " Gene capable of coding Acetohydroxy acid synthetase and its use, " Patent:JP 1993344893-A 1 12/27/93	E06111	It is mutated prephenate dehydratase
E06146		Acetohydroxy acid synthetase		E06825	Aspartokinase	Sugimoto, M.et al. " Mutant aspartokinase gene, " patent:JP 1994062866-A 1 03/08/94
E06826		It is mutated aspartokinase alpha subunit	Sugimoto, M.et al. " Mutant aspartokinase gene, " patent:JP 1994062866-A 1 03/08/94	E06825	Aspartokinase
E06826		It is mutated aspartokinase alpha subunit		E06827	It is mutated aspartokinase alpha subunit	Sugimoto, M.et al. " Mutant aspartokinase gene, " patent:JP 1994062866-A 1 03/08/94
E07701	secY		Honno, N.et al. " Gene DNA participating in integration of membraneous protein to membrane, " Patent:JP 1994169780-A 1 06/21/94	E06827	It is mutated aspartokinase alpha subunit
E07701	secY			E08177	Aspartokinase	Sato, Y.et al. " Genetic DNA capable of coding Aspartokinase released from feedback inhibition and its utilization, " Patent:JP 1994261766-A 1 09/20/94
E08178, E08179, E08180, E08181, E08182		Feedback inhibition-release aspartokinase	Sato, Y.et al. " Genetic DNA capable of coding Aspartokinase released from feedback inhibition and its utilization, " Patent:JP 1994261766-A 1 09/20/94	E08177	Aspartokinase
E08178, E08179, E08180, E08181, E08182		Feedback inhibition-release aspartokinase		E08232	Acetohydroxy acid isomeroreductase	Inui, M.et al. " Gene DNA coding acetohydroxy acid isomeroreductase, " Patent:JP 1994277067-A 1 10/04/94
E08234	secE		Asai, Y.et al. " Gene DNA coding for translocation machinery of protein, " Patent:JP 1994277073-A 1 10/04/94	E08232	Acetohydroxy acid isomeroreductase
E08234	secE			E08643	FT transaminase and dethiobiotin synthetase promoter region	Hatakeyama, K.et al. " DNA fragment having promoter function in coryneform bacterium, " Patent:JP 1995031476-A 1 02/03/95
E08646		Biotin synzyme	Hatakeyama, K.et al. " DNA fragment having promoter function in coryneform bacterium, " Patent:JP 1995031476-A 1 02/03/95	E08643	FT transaminase and dethiobiotin synthetase promoter region

GenBank^TMSearching number

Gene Name

Gene function

Bibliography

E08649		Aspartase	Kohama, K.et al " DNA fragment having promoter function in coryneform bacterium, " Patent:JP 1995031478-A 1 02/03/95
E08649		Aspartase		E08900		Dihydro 2, dipicolimic acid 2 reductase	Madori, M.et al. " DNA fragment containing gene coding Dihydrodipicolinate acid reductase and utilization thereof, " Patent:JP 1995075578-A 103/20/95
E08901		Diaminapimelate decarboxylase	Madori, M.et al. " DNA fragment containing gene coding Diaminopimelic acid decarboxylase and utilization thereof, " Patent:JP 1995075579-A 1 03/20/95	E08900		Dihydro 2, dipicolimic acid 2 reductase
E08901		Diaminapimelate decarboxylase		E12594		Serine hydroxymethylase	Hatakeyama, K.et al. " Production of L-trypophan, " Patent:JP 1997028391-A 1 02/04/97
E12760, E12759, E12758		Transposase	Moriya, M.et al. " Amplification of gene using artificial transposon, " Patent:JP 1997070291-A 03/18/97	E12594		Serine hydroxymethylase
E12760, E12759, E12758		Transposase		E12764		Arginyl-tRNA synthetase；Diaminapimelate decarboxylase	Moriya, M.et al. " Amplification of gene using artificial transposon, " Patent:JP 1997070291-A 03/18/97
E12767		Dihydro 2, dipicolimic acid 2 synzyme	Moriya, M.et al. " Amplification of gene using artificial transposon, " Patent:JP 1997070291-A 03/18/97	E12764		Arginyl-tRNA synthetase；Diaminapimelate decarboxylase
E12767		Dihydro 2, dipicolimic acid 2 synzyme		E12770		Aspartokinase	Moriya, M.et al. " Amplification of gene using artificial transposon, " Patent:JP 1997070291-A 03/18/97
E12773		Dihydro 2, dipicolimic acid 2 reductase	Moriya, M.et al. " Amplification of gene using artificial transposon, " Patent:JP 1997070291-A 03/18/97	E12770		Aspartokinase
E12773		Dihydro 2, dipicolimic acid 2 reductase		E13655		Glucose-6-phosphate dehydrogenase (G6PD)	Hatakeyama, K, et al. " Glucose-6-phosphate dehydrogenase and DNA capable of coding the same, " Patent:JP 1997224661-A 1 09/02/97
L01508	IlvA	Threonine dehydratase	Moeckel, B.et al. " Functional and structural analysis of the threonine dehydratase of Corynebacterium glutamicum, " J.Bacteriol., 174:8065-8072 (1992)	E13655		Glucose-6-phosphate dehydrogenase (G6PD)
L01508	IlvA	Threonine dehydratase		L07603	EC 4.2.1.15	3- deoxidation-D-arabinose heptanone saccharic acid -7- phosphate synthase	Chen, C.et al. " The cloning and nucleotide sequence of Corynebacterium glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene; " FEMS Microbiol.Lett., 107:223-230 (1993)
L09232	IlvB；ilvN；ilvC	Acetohydroxy acid synthase large subunit；Acetohydroxy acid synthase small subunit；Acetohydroxy acid isomeroreductase	Keilhauer, C.et al. " Isoleucine synthesis in Corynebacterium glutamicum:molecular analysis of the ilvB-ilvN-ilvC operon; " J.Bacteriol, 175 (17): 5595- 5603 (1993)	L07603	EC 4.2.1.15

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	L18874	PtsM	Phosphoenolpyruvate sugar phosphotransferase	Fouet, A et al. " Bacillus subtilis sucrose-specific enzyme II of the

			Phosphotransferase system:expression in Escherichia coli and homology to enzymes II from enteric bacteria, " PNAS USA, 84 (24): 8773-8777 (1987)；Lee, J.K.et al. " Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence; " FEMS Microbiol.Lett., 119 (1-2): 137-145 (1994)
				L27123	aceB	Malate synthase	Lee, H-S.et al. " Molecular characterization of aceB; a gene encoding malate synthase in Corynebacterium glutamicum, " J.Microbiol.Biotechnol., 4 (4): 256-263 (1994)
L27126		Pyruvate kinase	Jetten, M.S.et al. " Structural and functional analysis of pyruvate kinase from Corynebacterium glutamicum; " Appl.Environ.Microbiol., 60 (7): 2501-2507 (1994)	L27123	aceB	Malate synthase
L27126		Pyruvate kinase		L28760	aceA	Isocitratase
L35906	dtxr	Diphtheria toxin repressor	Oguiza, J.A.et al. " Molecular cloning; DNA sequence analysis; and characterization of the Corynebacterium diphtheriae dtxR from Brevibacterium lactofermentum; " J.Bacteriol., 177 (2): 465-467 (1995)	L28760	aceA	Isocitratase
L35906	dtxr	Diphtheria toxin repressor		M13774		Prephenate dehydratase	Follettie, M.T.et al. " Molecular cloning and nucleotide sequence of the Corynebacterium glutamicum pheA gene, " J.Bacteriol., 167:695-702 (1986)
M16175	5S rRNA		Park, Y-H.et al. " 56 rRNA sequences of Phylogenetic analysis of the coryneform bacteria by, " J.Bacteriol., 169:1801-1806 (1987)	M13774		Prephenate dehydratase
M16175	5S rRNA			M16663	trpE	Anthranilate synthase, 5 ' ends	Sano, K.et al. " Structure and function of the trp operon control regions of Brevibacterium lactofermentum; a glutamic-acid-producing bacterium, " Gene, 52:191-200 (1987)
M16664	trpA	Tryptophan synthetase, 3 ' ends	Sano, K.et al. " Structure and function of the trp operon control regions of Brevibacterium lactofermentum; a glutamic-acid-producing bacterium, " Gene, 52:191-200 (1987)	M16663	trpE	Anthranilate synthase, 5 ' ends
M16664	trpA	Tryptophan synthetase, 3 ' ends		M25819		Phosphoenolpyruvate carboxylase	O ' Regan, M.et al. " Cloning and nucleotide sequence of the Phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032; " Gene, 77 (2): 237-251 (1989)
M85106		23S rRNA gene inserts	Roller, C.et al. " Gram-positive bacteria with a high DNA G+C content are characterized by a common insertion within their 23S rRNA genes; " J.Gen. Microbiol., 138:1167-1175 (1992)	M25819		Phosphoenolpyruvate carboxylase

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	M85107,		23S rRNA gene inserts	Roller, C.et al. " Gram-positive bacteria with a high DNA G+C content are

M85108			Characterized by a common insertion within their 23S rRNA genes, " J.Gen. Microbiol., 138:1167-1175 (1992)
M85108				M89931	aecD；brnQ；yhbw	Beta C-S lyase；Branched-chain amino acid absorbs carrier；The albumen yhbw of presumption	Rossol, I.et al. " The Corynebacterium glutamicum aecD gene encodes a C-S lyase with alpha; beta-elimination activity that degrades aminoethylcysteine; " J.Bacteriol., 174 (9): 2968-2977 (1992)；Tauch, A.et al. " 13032 is directed by the brnQ gene product of Isoleucine uptake in Corynebacterium glutamicum ATCC; " Arch.Microbiol., 169 (4): 303-312 (1998)
S59299	trp	Leading gene (promoter)	Herry, D.M.et al. " Cloning of the trp gene cluster from a tryptophan- hyperproducing strain of Corynebacterium glutamicum:identification of a mutation in the trp leader sequence; " Appl.Environ.Microbiol., 59 (3): 791-799 (1993)	M89931	aecD；brnQ；yhbw
S59299	trp	Leading gene (promoter)		U11545	trpD	Anthranilate phosphoribosyl transferase	O ' Gara, J.P.and Dunican, L.K. 21850 tpD gene. " Thesis of (1994) Complete nucleotide sequence of the Corynebacterium glutamicum ATCC; Microbiology Department; University College Galway, Ireland.
U13922	cglIM；cglIR；clgIIR	The II type 5- cytosine methyltransferase of supposition；The II type restriction enzyme of supposition；The I type or type III restriction enzyme of supposition	Schafer, A.et al. " 13032 and analysis of its role in intergeneric conjugation with Escherichia coli of Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC; " J.Bacteriol., 176 (23): 73 09-7319 (1994)；Schafer, A.et al. " The Corynebacterium glutamicum cglIM gene encoding a 5-cytosine in an McrBC- deficient Escherichia coli strain; " Gene, 203 (2): 95-101 (1997)	U11545	trpD	Anthranilate phosphoribosyl transferase
U13922	cglIM；cglIR；clgIIR			U14965	recA
U31224	ppx		Ankri, S.et al. " Mutations in the Corynebacterium glutamicumproline biosynthetic pathway:A natural bypass of the proA step; " J.Bacteriol., 178 (15): 4412-4419 (1996)	U14965	recA
U31224	ppx			U31225	proC	L-PROLINE: NADP+5- oxidoreducing enzyme	Ankri, S.et al. " Mutations in the Corynebacterium glutamicumproline biosynthetic pathway:A natural bypass of the proA step; " J.Bacteriol., 178 (15): 4412-4419 (1996)
U31230	obg；proB；unkdh	；Gamma Glutamyl kinase；Similar to the special 2- hydroxy acid dehydrogenase of D- isomers	Ankri, S.et al. " Mutations in the Corynebacterium glutamicumproline biosynthetic pathway:A natural bypass of the proA step; " J.Bacteriol., 178 (15): 4412-4419 (1996)	U31225	proC	L-PROLINE: NADP+5- oxidoreducing enzyme

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	U31281	bioB	Biotin synthase	Serebriiskii, I.G., " Two new members of the bio B superfamily:Cloning,

			Sequencing and expression of bio B genes of Methylobacillus flagellatum and Corynebacterium glutamicum, " Gene, 175:15-22 (1996)
				U35023	thtR；accBC	Thiosulfate transsulfurase；Acyl-CoA carboxylase	Jager, W.et al. " A Corynebacterium glutamicum gene encoding a two-domain protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins; " Arch.Microbiol., 166 (2)；76-82(1996)
U43535	cmr	Multi-drug resistance albumen	Jager, W.et al. " A Corynebacterium glutamicum gene conferring multidrug resistance in the heterologous host Escherichia coli; " J.Bacteriol., 179 (7): 2449-2451 (1997)	U35023	thtR；accBC	Thiosulfate transsulfurase；Acyl-CoA carboxylase
U43535	cmr	Multi-drug resistance albumen		U43536	clpB	Heat shock ATP- binding protein
U53587	aphA-3	3 ' 5 "-aminoglycoside phosphotransferases		U43536	clpB	Heat shock ATP- binding protein
U53587	aphA-3	3 ' 5 "-aminoglycoside phosphotransferases		U89648		The Corynebacterium glutamicum for participating in histidine biosynthesis does not identify sequence, partial sequence
X04960	trpA；trpB；trpC；trpD；trpE；trpG；trpL	Tryptophan operon	Matsui, K.et al. " Complete nucleotide and deduced amino acid sequences of the Brevibacterium lactofermentum tryptophan operon; " Nucleic Acids Res., 14 (24): 10113-10114 (1986)	U89648
X04960	trpA；trpB；trpC；trpD；trpE；trpG；trpL	Tryptophan operon		X07563	lys A	DAP decarboxylase (meso-diaminapimelate decarboxylase, EC 4.1.1.20)	Yeh, P.et al. " Nucleic sequence of the lysA gene of Corynebacterium glutamicum and possible mechanisms for modulation of its expression; " Mol. Gen.Genet., 212 (1): 112-119 (1988)
X14234	EC 4.1.1.31	Phosphoenolpyruvate carboxylase	Eikmanns, B.J.et al. " The Phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum:Molecular cloning; nucleotide sequence; and expression; " Mol.Gen.Genet., 218 (2): 330-339 (1989)；Lepiniec, L.et al. " Sorghum Phosphoenolpyruvate carboxylase gene family:structure; function and molecular evolution, " Plant.Mol.Biol., 21 (3): 487-502 (1993)	X07563	lys A
X14234	EC 4.1.1.31	Phosphoenolpyruvate carboxylase		X17313	fda	Fructose-bisphosphate aldolase	Von der Osteu, C.H.et al. " Molecular cloning; nucleotide sequence and fine- structural analysis of the Corynebacterium glutamicum fda gene:structural comparison of C.glutamicum fructose-1; 6-biphosphate aldolase to class I and class II aldolases, " Mol.Micr Obiol.,
X53993	dapA	L-2,3- dihydro 2, dipicolimic acid 2 synzyme (EC 4.2.1.52)	Bonnassie, S.et al. " Nucleic sequence of the dapA gene from Corynebacterium glutamicum, " Nucleic Acids Res., 18 (21): 6421 (1990)	X17313	fda	Fructose-bisphosphate aldolase

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	X54223		AttB- related locus	Cianciotto, N.et al. " DNA sequence homology between att B-related sites of

			Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP site of lambdacorynephage; " FEMS.Microbiol. Lett., 66:299-302 (1990)
				X54740	argS；lysA	Arginyl-tRNA synthetase；Diaminapimelate decarboxylase	Marcel, T.et al. " Nucleotide sequence and organization of the upstream region of the Corynebacterium glutamicum lysA gene; " Mol.Microbiol., 4 (11): 1819- 1830 (1990)
X55994	trpL；trpE	The leader peptide of supposition；Anthranilate synthase ingredient 1	Heery, D.M.et al. " Nucleotide sequence of the Corynebacterium glutamicum trpE gene, " Nucleic Acids Res., 18 (23): 7138 (1990)	X54740	argS；lysA	Arginyl-tRNA synthetase；Diaminapimelate decarboxylase
X55994	trpL；trpE			X56037	thrC	Threonine synthase	Han, K.S.et al. " The molecular structure of the Corynebacterium glutamicum threonine synthase gene, " Mol.Microbiol., 4 (10): 1693-1702 (1990)
X56075	AttB- related locus	Attachment site	Cianciotto, N.et al. " DNA sequence homology between att B-related sites of Corynebacterium diphtheriae; Corynebacterium ulcerans; Corynebacterium glutamicum; and the attP site of lambdacorynephage; " FEMS.Microbiol, Lett., 66:299-302 (1990)	X56037	thrC	Threonine synthase
X56075	AttB- related locus	Attachment site		X57226	lysC-alpha；lysC-beta； asd	Aspartokinase-alpha subunit；Aspartokinase-beta subunit；Aspartic acid beta semialdehyde dehydrogenase	Kalinowski, J.et al. " Genetic and biochemical analysis of the Aspartokinase from Corynebacterium glutamicum; " Mol.Microbiol., 5 (5): 1197-1204 (1991)；Kalinowski, J.et al. " Aspartokinase genes lysC alpha and lysC beta overlap and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum; " Mol.Gen.Genet., 224 (3): 317-324 (1990)
X59403	gap；pgk；tpi	Glyceraldehyde-3-phosphate；Phosphoglyceric kinase；Triose-phosphate isomerase	Eikmanns, B.J. " Identification; sequence analysis; and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase; 3-phosphoglycerate kinase; and triosephosphate isomeras, " J.Bacteri Ol., 174 (19): 6076-6086 (1992)	X57226	lysC-alpha；lysC-beta； asd
X59403	gap；pgk；tpi			X59404	gdh	Glutamte dehydrogenase	Bormann, E.R.et al. " Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase; " Mol.Microbiol., 6 (3): 317-326 (1992)
X60312	lysI	L-lysine permease	Seep-Feldhaus, A.H.et al. " Molecular analysis of the Corynebacterium glutamicum lysl gene involved in lysine uptake; " Mol.Microbiol., 5 (12): 2995- 3005 (1991)	X59404	gdh	Glutamte dehydrogenase

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	X66078	cop1	Ps1 albumen	Joliff, G.et al. " Cloning and nucleotide sequence of the csp1 gene encoding

			PS1,85 complex of one of the two major secreted proteins of Corynebacterium glutamicum:The deduced N-terminal region of PS1 is similar to the Mycobacterium antigen; " Mol.Microbiol., 6 (16): 2349-2362 (1992)
				X66112	glt	Citrate synthase	Eikmanns, B.J.et al. " Cloning sequence; expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase; " Microbiol., (1994) 140:1817-1828
X67737	dapB	Dihydro 2, dipicolimic acid 2 reductase		X66112	glt	Citrate synthase
X67737	dapB	Dihydro 2, dipicolimic acid 2 reductase		X69103	csp2	S-layer proteins PS2	Peyret, J.L.et al. " Characterization of the cspB gene encoding PS2; an ordered surface-layer protein in Corynebacterium glutamicum; " Mol.Microbiol., 9 (1): 97-109 (1993)
X69104		IS3 correlation is inserted into the factor	Bonamy, C.et al. " Identification of IS1206; a Corynebacterium glutamicum IS3-related insertion sequence and phylogenetic analysis; " Mol.Microbiol., 14 (3): 571-581 (1994)	X69103	csp2	S-layer proteins PS2
X69104		IS3 correlation is inserted into the factor		X70959	leuA	Isopropylmalate synthase	Patek, M.et al. " Leucine synthesis in Corynebacterium glutamicum:enzyme activities; structure of leuA; and effect of leuA inactivation on lysine synthesis; " Appl.Environ.Microbiol., 60 (1): 133-140 (1994)
X71489	icd	Isocitric dehydrogenase (NADP+)	Eikmanns, B.J.et al. " Cloning sequence analysis; expression; and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme; " J.Bacteriol., 177 (3): 774-782 (1995)	X70959	leuA	Isopropylmalate synthase
X71489	icd	Isocitric dehydrogenase (NADP+)		X72855	GDHA	Glutamte dehydrogenase (NADP+)
X75083, X70584	mtrA	5-methyl tryptophan resistance	Heery, D.M.et al. " A sequence from a tryptophan-hyperproducing strain of Corynebacterium glutamicum encoding resistance to 5-methyltryptophan; " Biochem.Biophys.Res.Commun., 201 (3): 1255-1262 (1994)	X72855	GDHA	Glutamte dehydrogenase (NADP+)
X75083, X70584	mtrA	5-methyl tryptophan resistance		X75085	recA		Fitzpatrick, R.et al. " Construction and characterization of recA mutant strains of Corynebacterium glutamicum and Brevibacterium lactofermentum; " Appl. Microbiol.Biotechnol., 42 (4): 575-580 (1994)
X75504	aceA；thiX	Part isocitratase；	Reinscheid, D.J.et al. " Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme; " J. Bacteriol., 176 (12): 3474-3483 (1994)	X75085	recA
X75504	aceA；thiX	Part isocitratase；		X76875		ATP enzyme beta- subunit	Ludwig, W.et al. " Phylogenetic relationships of bacteria based on comparative sequence analysis of elongation factor Tu and ATP-synthase beta-subunit genes; " Antonie Van Leeuwenhoek, 64:285-305 (1993)
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	X76875		ATP enzyme beta- subunit
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	X77034	tuf	Extension factor T u	Ludwig, W.et al. " Phylogenetic relationships of bacteria based on comparative

			Sequence analysis of elongation factor Tu and ATP-synthase beta-subunit genes, " Antonie Van Leeuwenhoek, 64:285-305 (1993)
				X77384	recA		Billman-Jacobe, H. " Nucleotide sequence of a recA gene from Corynebacterium glutamicum, " DNA Seq., 4 (6): 403-404 (1994)
X78491	aceB	Malate synthase	Reinscheid, D.J.et al. " Malate synthase from Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase:sequence analysis; " Microbiology, 140:3099-3108 (1994)	X77384	recA
X78491	aceB	Malate synthase		X80629	16S rDNA	16S rRNA	Rainey, F.A.et al. " Phylogenetic analysis of the genera Rhodococcus and Norcardia and evidence for the evolutionary origin of the genus Norcardia within the radiation of Rhodococcus species; " Microbiol., (1995) 141:523-528
X81191	gluA；gluB；gluC； gluD	Glutamic acid absorption system	Kronemeyer, W.et al. " Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum; " J.Bacteriol., 177 (5): 1152-1158 (1995)	X80629	16S rDNA	16S rRNA
X81191	gluA；gluB；gluC； gluD	Glutamic acid absorption system		X81379	dapE	Succinyldiaminopimelate desuccinylase	Wehrmann, A.et al. " Analysis of different DNA fragments of Corynebacterium glutamicum complementing dapE of Escherichia coli; " Microbiology, 40:3349-56 (1994)
X82061	16S rDNA	16S rRNA	Ruimy, R.et al. " Phylogeny of the genus Corynebacterium dednced from analyses of small-subunit ribosomal DNA sequences; " Int.J.Syst.Bacteriol., 45 (4): 740-746 (1995)	X81379	dapE	Succinyldiaminopimelate desuccinylase
X82061	16S rDNA	16S rRNA		X82928	asd；lysC	Aspartate-semialdehyde dehydrogenase；	Serebrijski, I.et al. " Multicopy suppression by asd gene and osmotic stress- dependent complementation by heterologous proA in proA mutants; " J. Bacteriol., 177 (24): 7255-7260 (1995)
X82929	proA	Gamma- glutamyl phosphate reductase	Serebrijski, I.et al. " Multicopy suppression by asd gene and osmotic stress- dependent complementation by heterologous proA in proA mutants; " J. Bacteriol., 177 (24): 7255-7260 (1995)	X82928	asd；lysC	Aspartate-semialdehyde dehydrogenase；
X82929	proA	Gamma- glutamyl phosphate reductase		X84257	16S rDNA	16S rRNA	Pascual, C.et al. " Phylogenetic analysis of the genus Corynebacterium based on 16S rRNA gene sequences; " Int.J.Syst.Bacteriol., 45 (4): 724-728 (1995)
X85965	aroP；dapE	Aromatic amino acid permease；	Wehrmann, A.et al. " Functional analysis of sequences abjacent to dapE of Corynebacterium glutamicumproline reveals the presence of aroP; which encodes the aromatic amino acid transporter; " J.Bacteriol., 177 (20): 5991- 5993 (1995)	X84257	16S rDNA	16S rRNA

GenBank^TMSearching number

Gene Name

Gene function

Bibliography

X86157	argB；argC；argD； argF；argJ	Acetylglutamate kinase；N- acetyl-gamma- glutamy-phosphoric acid reduction enzyme；Acetyl-ornithine transaminase；Ornithine transcarbamylase；Glutamic acid N-transacetylase	Sakanyan, V.et al. " Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum:enzyme evolution in the early steps of the arginine pathway; " Microbiology, 142:99-108 (1996)
X86157	argB；argC；argD； argF；argJ			X89084	pta；ackA	Phosphate acetyltransferase；Acetokinase	Reinscheid, D.J.et al. " Cloning; sequence analysis; expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase; " Microbiology, 145:503-513 (1999)
X89850	attB	Attachment site	Le Marrec, C.et al. " Genetic characterization of site-specific integration functions of phi AAU2 infecting " Arthrobacter aureus C70; " J.Bacteriol., 178 (7): 1996-2004 (1996)	X89084	pta；ackA	Phosphate acetyltransferase；Acetokinase
X89850	attB	Attachment site		X90356		Promoter fragment F1	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)
X90357		Promoter fragment F2	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)	X90356		Promoter fragment F1
X90357		Promoter fragment F2		X90358		Promoter fragment F10	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)
X90359		Promoter fragment F13	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)	X90358		Promoter fragment F10
X90359		Promoter fragment F13		X90360		Promoter fragment F22	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)
X90361		Promoter fragment F34	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)	X90360		Promoter fragment F22
X90361		Promoter fragment F34		X90362		Promoter fragment F37	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	X90363		Promoter fragment F45	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)

X90364		Promoter fragment F64	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)
X90364		Promoter fragment F64		X90365		Promoter fragment F75	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)
X90366		Promoter fragment PF101	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)	X90365		Promoter fragment F75
X90366		Promoter fragment PF101		X90367		Promoter fragment PF104	Pate k, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)
X90368		Promoter fragment PF109	Patek, M.et al. " Promoters from Corynebacterium glutamicum:cloning; molecular analysis and search for a consensus motif, " Microbiology, 142:1297-1309 (1996)	X90367		Promoter fragment PF104
X90368		Promoter fragment PF109		X93513	amt	Ammonia movement system	Siewe, R.M.et al. " Functional and genetic characterization of the (methyl) ammonium uptake carrier of Corynebacterium glutamicum; " J.Biol.Chem., 271 (10): 5398-5403 (1996)
X93514	betP	Glycinebetaine movement system	Peter, H.et al. " Isolation; characterization; and expression of the Corynebacterium glutamicum betP gene; encoding the transport system for the compatible solute glycine betaine; " J.Bacteriol., 178 (17): 5229-5234 (1996)	X93513	amt	Ammonia movement system
X93514	betP	Glycinebetaine movement system		X95649	orf4		Patek, M.et al. " Identification and transcriptional analysis of the dapB-ORF2- dapA-ORF4 operon of Corynebacterium glutamicum; encoding two enzymes involved in L-lysine synthesis; " Biotechnol.Lett., (1997) 19:1113-1117
X96471	lysE；lysG	Lysine exports albumen；Lysine exports regulatory protein	Vrljic, M.et al. " A new type of transporter with a new type of cellular function:L-lysine export from Corynebacterium glutamicum; " Mol. Microbiol., 22 (5): 815-826 (1996)	X95649	orf4

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	X96580	panB；panC；xylB	3- methyl -2- oxy butyrate hydroxymethyl transferases；Pantothenic acid-beta- alanine ligase；Xylulokinase	Sahm, H.et al. " D-pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction; " Appl.Environ.Microbiol., 65 (5): 1973-1979 (1999)

X96962		Insetion sequence IS1207 and transposase
X96962		Insetion sequence IS1207 and transposase		X99289		Elongation factors P	Ramos, A.et al. " Cloning; sequencing and expression of the gene encoding elongation factor P in the amino-acid producer Brevibacterium lactofermentum (Corynebacterium glutamicum ATCC 13869); " Gene, 198:217-222 (1997)
Y00140	thrB	Homoserine kinase	Mateos, L.M.et al. " Nucleotide sequence of the homoserine kinase (thrB) gene of the Brevibacterium lactofermentum; " Nucleic Acids Res., 15 (9): 3922 (1987)	X99289		Elongation factors P
Y00140	thrB	Homoserine kinase		Y00151	ddh	Meso-diaminopimelic acid D- dehydrogenase (EC 1.4.1.16)	Ishino, S.et al. " Nucleotide sequence of the meso-diaminopimelate D- dehydrogenase gene from Corynebacterium glutamicum; " Nucleic Acids Res., 15 (9): 3917 (1987)
Y00476	thrA	Homoserine dehydrogenase	Mateos, L.M.et al. " Nucleotide sequence of the homoserine dehydrogenase (thrA) gene of the Brevibacterium lactofermentum; " Nucleic Acids Res., 15 (24): 10598 (1987)	Y00151	ddh	Meso-diaminopimelic acid D- dehydrogenase (EC 1.4.1.16)
Y00476	thrA	Homoserine dehydrogenase		Y00546	hom；thrB	Homoserine dehydrogenase；Homoserine kinase	Peoples, O.P.et al. " Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon; " Mol.Microbiol., 2 (1): 63-72 (1988)
Y08964	murC；ftsQ/divD；ftsZ	UPD-N- acetylmuramic acid-alanine ligase；Division starting albumen or Cyclin；Cyclin	Honrubia, M.P.et al. " Identification; characterization; and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum; " Mol. Gen.Genet., 259 (1): 97-104 (1998)	Y00546	hom；thrB	Homoserine dehydrogenase；Homoserine kinase
Y08964	murC；ftsQ/divD；ftsZ			Y09163	putP	High-affinity proline transporter systems	Peter, H.et al. " Isolation of the putP gene of Corynebacterium glutamicumproline and characterization of a low-affinity uptake system for compatible solutes; " Arch.Microbiol., 168 (2): 143-151 (1997)
Y09548	pyc	Pyruvate carboxylase	Peters-Wendisch, P.G.et al. " Pyruvate carboxylase from Corynebacterium glutamicum:characterization; expression and inactivation of the pyc gene; " Microbiology, 144:915-927 (1998)	Y09163	putP	High-affinity proline transporter systems
Y09548	pyc	Pyruvate carboxylase		Y09578	leuB	3-Isopropylmalate dehydrogenase	Patek, M.et al. " Analysis of the leuB gene from Corynebacterium glutamicum, " Appl.Microbiol.Biotechnol., 50 (1): 42-47 (1998)
Y12472		Bacteriophage Phi-16 attachment site	Moreau, S.et al. " Site-specific integration of corynephage Phi-16:The construction of an integration vector, " Microbiol., 145:539-548 (1999)	Y09578	leuB	3-Isopropylmalate dehydrogenase

GenBank^TMSearching number	Gene Name	Gene function	Bibliography
GenBank^TMSearching number	Gene Name	Gene function	Bibliography	Y12537	proP	Proline/ectoine absorption system albumen	Peter, H.et al. " Corynebacterium glutamicum is equipped with four secondary carriers for compatible solutes:Identification; sequencing; and characterization of the proline/ectoine uptake system; ProP, and the ectoine/proline/glycine

			Betaine carrier, EctP, " J.Bacteriol., 180 (22): 6005-6012 (1998)
				Y13221	glnA	Glutamine synthelase I	Jakoby, M.et al. " Isolation of Corynebacterium glutamicum glnA gene encoding glutamine synthetase I, " FEMS Microbiol.Lett., 154 (1): 81-88 (1997)
Y16642	lpd	Dihydrolipoamide dehydrogenase		Y13221	glnA	Glutamine synthelase I
Y16642	lpd	Dihydrolipoamide dehydrogenase		Y18059		Bar bacterium bacteriophage 304L attachment site	Moreau, S.et al. " Analysis of the integration functions of &phi；304L:An integrase module among corynephages, " Virology, 255 (1): 150-159 (1999)
Z21501	argS；lysA	Arginyl-tRNA synthetase；Diaminapimelate decarboxylase (part)	Oguiza, J.A.et al. " A gene encoding arginyl-tRNA synthetase is located in the upstream region of the lysA gene in Brevibacterium lactofermentum:Regulation of argS-lysA cluster expression by arginine; " J. Bacteriol., 175 (22): 7356-7362 (1993)	Y18059		Bar bacterium bacteriophage 304L attachment site
Z21501	argS；lysA			Z21502	dapA；dapB	Dihydro 2, dipicolimic acid 2 synthase；Dihydro 2, dipicolimic acid 2 reductase	Pisabarro, A.et al. " A cluster of three genes (dapA; orf2; and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate reductase; and a third polypeptide of unknown function; " J.Bacteriol., 175 (9): 2743-2749 (1993)
Z29563	thrC	Threonine synthase	Malumbres, M.et al. " Analysis and expression of the thrC gene of the encoded threonine synthase, " Appl.Environ.Microbiol., 60 (7) 2209-2219 (1994)	Z21502	dapA；dapB
Z29563	thrC	Threonine synthase		Z46753	16S rDNA	16S ribosomal RNA gene
Z49822	sigA	The SigA sigma factor	Oguiza, J.A.et al " Multiple sigma factor genes in Brevibacterium lactofermentum:Characterization of sigA and sigB; " J.Bacteriol., 178 (2): 550- 553 (1996)	Z46753	16S rDNA	16S ribosomal RNA gene
Z49822	sigA	The SigA sigma factor		Z49823	galE；dtxR	UDP- galactolipin 4- epimerase catalytic activity；Diphtheria toxin regulatory protein	Oguiza, J.A.et al " The galE gene encoding the UDP-galactose 4-epimerase of Brevibacterium lactofermentum is coupled transcriptionally to the dmdR gene; " Gene, 177:103-107 (1996)
Z49824	orf1；sigB	；The SigB sigma factor	Oguiza, J.A.et al " Multiple sigma factor genes in Brevibacterium lactofermentum:Characterization of sigA and sigB; " J.Bacteriol., 178 (2): 550- 553 (1996)	Z49823	galE；dtxR
Z49824	orf1；sigB	；The SigB sigma factor		Z66534		Transposase	Correia, A.et al. " Cloning and characterization of an IS-like element present in the genome of Brevibacterium lactofermentum ATCC 13869; " Gene, 170 (1): 91-94 (1996)

¹The gene order has disclosed in listed bibliography.But the sequence that the present invention obtains is obviously long compared with sequence is disclosed.Speculate disclosed ATG code of sequence mistake, therefore a segment in only actual coding area.

Table 3: it can be used for implementing bar bacterium and Brevibacterium flavum strain of the invention

Generic name	Kind name	ATCC	FERM	NRRL	CECT	NCIMB	CBS	NCTC	DSMZ
Generic name	Kind name	ATCC	FERM	NRRL	CECT	NCIMB	CBS	NCTC	DSMZ	Brevibacterium	Brevibacterium ammoniagene	21054
Brevibacterium	Brevibacterium ammoniagene	19350								Brevibacterium	Brevibacterium ammoniagene	21054
Brevibacterium	Brevibacterium ammoniagene	19350								Brevibacterium	Brevibacterium ammoniagene	19351
Brevibacterium	Brevibacterium ammoniagene	19352								Brevibacterium	Brevibacterium ammoniagene	19351
Brevibacterium	Brevibacterium ammoniagene	19352								Brevibacterium	Brevibacterium ammoniagene	19353
Brevibacterium	Brevibacterium ammoniagene	19354								Brevibacterium	Brevibacterium ammoniagene	19353
Brevibacterium	Brevibacterium ammoniagene	19354								Brevibacterium	Brevibacterium ammoniagene	19355
Brevibacterium	Brevibacterium ammoniagene	19356								Brevibacterium	Brevibacterium ammoniagene	19355
Brevibacterium	Brevibacterium ammoniagene	19356								Brevibacterium	Brevibacterium ammoniagene	21055
Brevibacterium	Brevibacterium ammoniagene	21077								Brevibacterium	Brevibacterium ammoniagene	21055
Brevibacterium	Brevibacterium ammoniagene	21077								Brevibacterium	Brevibacterium ammoniagene	21553
Brevibacterium	Brevibacterium ammoniagene	21580								Brevibacterium	Brevibacterium ammoniagene	21553
Brevibacterium	Brevibacterium ammoniagene	21580								Brevibacterium	Brevibacterium ammoniagene	39101
Brevibacterium	butanicum	21196								Brevibacterium	Brevibacterium ammoniagene	39101
Brevibacterium	butanicum	21196								Brevibacterium	Branch brevibacterium	21792	P928
Brevibacterium	Brevibacterium flavum	21474								Brevibacterium	Branch brevibacterium	21792	P928
Brevibacterium	Brevibacterium flavum	21474								Brevibacterium	Brevibacterium flavum	21129
Brevibacterium	Brevibacterium flavum	21518								Brevibacterium	Brevibacterium flavum	21129
Brevibacterium	Brevibacterium flavum	21518								Brevibacterium	Brevibacterium flavum			B11474
Brevibacterium	Brevibacterium flavum			B11472						Brevibacterium	Brevibacterium flavum			B11474
Brevibacterium	Brevibacterium flavum			B11472						Brevibacterium	Brevibacterium flavum	21127
Brevibacterium	Brevibacterium flavum	21128								Brevibacterium	Brevibacterium flavum	21127
Brevibacterium	Brevibacterium flavum	21128								Brevibacterium	Brevibacterium flavum	21427
Brevibacterium	Brevibacterium flavum	21475								Brevibacterium	Brevibacterium flavum	21427
Brevibacterium	Brevibacterium flavum	21475								Brevibacterium	Brevibacterium flavum	21517
Brevibacterium	Brevibacterium flavum	21528								Brevibacterium	Brevibacterium flavum	21517
Brevibacterium	Brevibacterium flavum	21528								Brevibacterium	Brevibacterium flavum	21529
Brevibacterium	Brevibacterium flavum			B11477						Brevibacterium	Brevibacterium flavum	21529
Brevibacterium	Brevibacterium flavum			B11477						Brevibacterium	Brevibacterium flavum			B11478
Brevibacterium	Brevibacterium flavum	21127								Brevibacterium	Brevibacterium flavum			B11478
Brevibacterium	Brevibacterium flavum	21127								Brevibacterium	Brevibacterium flavum			B11474
Brevibacterium	Xi Shi brevibacterium	15527								Brevibacterium	Brevibacterium flavum			B11474
Brevibacterium	Xi Shi brevibacterium	15527								Brevibacterium	Ketoglutaric acid brevibacterium	21004
Brevibacterium	Ketoglutaric acid brevibacterium	21089								Brevibacterium	Ketoglutaric acid brevibacterium	21004
Brevibacterium	Ketoglutaric acid brevibacterium	21089								Brevibacterium	ketosoreductum	21914
Brevibacterium	Brevibacterium				70					Brevibacterium	ketosoreductum	21914
Brevibacterium	Brevibacterium				70					Brevibacterium	Brevibacterium				74
Brevibacterium	Brevibacterium				77					Brevibacterium	Brevibacterium				74
Brevibacterium	Brevibacterium				77					Brevibacterium	Brevibacterium	21798
Brevibacterium	Brevibacterium	21799								Brevibacterium	Brevibacterium	21798
Brevibacterium	Brevibacterium	21799								Brevibacterium	Brevibacterium	21800
Brevibacterium	Brevibacterium	21801								Brevibacterium	Brevibacterium	21800
Brevibacterium	Brevibacterium	21801								Brevibacterium	Brevibacterium			B11470
Brevibacterium	Brevibacterium			B11471						Brevibacterium	Brevibacterium			B11470
Brevibacterium	Brevibacterium			B11471						Brevibacterium	Brevibacterium	21086
Brevibacterium	Brevibacterium	21420								Brevibacterium	Brevibacterium	21086

Brevibacterium	Brevibacterium	21086
Brevibacterium	Brevibacterium	21086				Brevibacterium	Brevibacterium	31269
Brevibacterium	Extension brevibacterium	9174				Brevibacterium	Brevibacterium	31269
Brevibacterium	Extension brevibacterium	9174				Brevibacterium	Extension brevibacterium	19391
Brevibacterium	Extension brevibacterium	8377				Brevibacterium	Extension brevibacterium	19391
Brevibacterium	Extension brevibacterium	8377				Brevibacterium	Solve paraffin brevibacterium			11160
Brevibacterium	Kind			717.73		Brevibacterium	Solve paraffin brevibacterium			11160
Brevibacterium	Kind			717.73		Brevibacterium	Kind				717.73
Brevibacterium	Kind	14604				Brevibacterium	Kind				717.73
Brevibacterium	Kind	14604				Brevibacterium	Kind	21860
Brevibacterium	Kind	21864				Brevibacterium	Kind	21860
Brevibacterium	Kind	21864				Brevibacterium	Kind	21865
Brevibacterium	Kind	21866				Brevibacterium	Kind	21865
Brevibacterium	Kind	21866				Brevibacterium	Kind	19240
Bar bacterium	Corynebacterium acctoacidophlum	21476				Brevibacterium	Kind	19240
Bar bacterium	Corynebacterium acctoacidophlum	21476				Bar bacterium	Corynebacterium acctoacidophlum	13870
Bar bacterium	Vinegar paddy bar bacterium		B11473			Bar bacterium	Corynebacterium acctoacidophlum	13870
Bar bacterium	Vinegar paddy bar bacterium		B11473			Bar bacterium	Vinegar paddy bar bacterium		B11475
Bar bacterium	Vinegar paddy bar bacterium	15806				Bar bacterium	Vinegar paddy bar bacterium		B11475
Bar bacterium	Vinegar paddy bar bacterium	15806				Bar bacterium	Vinegar paddy bar bacterium	21491
Bar bacterium	Vinegar paddy bar bacterium	31270				Bar bacterium	Vinegar paddy bar bacterium	21491
Bar bacterium	Vinegar paddy bar bacterium	31270				Bar bacterium	Thermophilic acetyl bar bacterium		B3671
Bar bacterium	Corynebacterium ammoniagenes	6872			2399	Bar bacterium	Thermophilic acetyl bar bacterium		B3671
Bar bacterium	Corynebacterium ammoniagenes	6872			2399	Bar bacterium	Corynebacterium ammoniagenes	15511
Bar bacterium	fujiokense	21496				Bar bacterium	Corynebacterium ammoniagenes	15511
Bar bacterium	fujiokense	21496				Bar bacterium	Corynebacterium glutamicum	14067
Bar bacterium	Corynebacterium glutamicum	39137				Bar bacterium	Corynebacterium glutamicum	14067
Bar bacterium	Corynebacterium glutamicum	39137				Bar bacterium	Corynebacterium glutamicum	21254
Bar bacterium	Corynebacterium glutamicum	21255				Bar bacterium	Corynebacterium glutamicum	21254
Bar bacterium	Corynebacterium glutamicum	21255				Bar bacterium	Corynebacterium glutamicum	31830
Bar bacterium	Corynebacterium glutamicum	13032				Bar bacterium	Corynebacterium glutamicum	31830
Bar bacterium	Corynebacterium glutamicum	13032				Bar bacterium	Corynebacterium glutamicum	14305
Bar bacterium	Corynebacterium glutamicum	15455				Bar bacterium	Corynebacterium glutamicum	14305
Bar bacterium	Corynebacterium glutamicum	15455				Bar bacterium	Corynebacterium glutamicum	13058
Bar bacterium	Corynebacterium glutamicum	13059				Bar bacterium	Corynebacterium glutamicum	13058
Bar bacterium	Corynebacterium glutamicum	13059				Bar bacterium	Corynebacterium glutamicum	13060
Bar bacterium	Corynebacterium glutamicum	21492				Bar bacterium	Corynebacterium glutamicum	13060
Bar bacterium	Corynebacterium glutamicum	21492				Bar bacterium	Corynebacterium glutamicum	21513
Bar bacterium	Corynebacterium glutamicum	21526				Bar bacterium	Corynebacterium glutamicum	21513
Bar bacterium	Corynebacterium glutamicum	21526				Bar bacterium	Corynebacterium glutamicum	21543
Bar bacterium	Corynebacterium glutamicum	13287				Bar bacterium	Corynebacterium glutamicum	21543
Bar bacterium	Corynebacterium glutamicum	13287				Bar bacterium	Corynebacterium glutamicum	21851
Bar bacterium	Corynebacterium glutamicum	21253				Bar bacterium	Corynebacterium glutamicum	21851
Bar bacterium	Corynebacterium glutamicum	21253				Bar bacterium	Corynebacterium glutamicum	21514
Bar bacterium	Corynebacterium glutamicum	21516				Bar bacterium	Corynebacterium glutamicum	21514
Bar bacterium	Corynebacterium glutamicum	21516				Bar bacterium	Corynebacterium glutamicum	21299
Bar bacterium	Corynebacterium glutamicum	21300				Bar bacterium	Corynebacterium glutamicum	21299
Bar bacterium	Corynebacterium glutamicum	21300				Bar bacterium	Corynebacterium glutamicum	39684
Bar bacterium	Corynebacterium glutamicum	21488				Bar bacterium	Corynebacterium glutamicum	39684
Bar bacterium	Corynebacterium glutamicum	21488				Bar bacterium	Corynebacterium glutamicum	21649

Bar bacterium	Corynebacterium glutamicum	21650
Bar bacterium	Corynebacterium glutamicum	21650			Bar bacterium	Corynebacterium glutamicum	19223
Bar bacterium	Corynebacterium glutamicum	13869			Bar bacterium	Corynebacterium glutamicum	19223
Bar bacterium	Corynebacterium glutamicum	13869			Bar bacterium	Corynebacterium glutamicum	21157
Bar bacterium	Corynebacterium glutamicum	21158			Bar bacterium	Corynebacterium glutamicum	21157
Bar bacterium	Corynebacterium glutamicum	21158			Bar bacterium	Corynebacterium glutamicum	21159
Bar bacterium	Corynebacterium glutamicum	21355			Bar bacterium	Corynebacterium glutamicum	21159
Bar bacterium	Corynebacterium glutamicum	21355			Bar bacterium	Corynebacterium glutamicum	31808
Bar bacterium	Corynebacterium glutamicum	21674			Bar bacterium	Corynebacterium glutamicum	31808
Bar bacterium	Corynebacterium glutamicum	21674			Bar bacterium	Corynebacterium glutamicum	21562
Bar bacterium	Corynebacterium glutamicum	21563			Bar bacterium	Corynebacterium glutamicum	21562
Bar bacterium	Corynebacterium glutamicum	21563			Bar bacterium	Corynebacterium glutamicum	21564
Bar bacterium	Corynebacterium glutamicum	21565			Bar bacterium	Corynebacterium glutamicum	21564
Bar bacterium	Corynebacterium glutamicum	21565			Bar bacterium	Corynebacterium glutamicum	21566
Bar bacterium	Corynebacterium glutamicum	21567			Bar bacterium	Corynebacterium glutamicum	21566
Bar bacterium	Corynebacterium glutamicum	21567			Bar bacterium	Corynebacterium glutamicum	21568
Bar bacterium	Corynebacterium glutamicum	21569			Bar bacterium	Corynebacterium glutamicum	21568
Bar bacterium	Corynebacterium glutamicum	21569			Bar bacterium	Corynebacterium glutamicum	21570
Bar bacterium	Corynebacterium glutamicum	21571			Bar bacterium	Corynebacterium glutamicum	21570
Bar bacterium	Corynebacterium glutamicum	21571			Bar bacterium	Corynebacterium glutamicum	21572
Bar bacterium	Corynebacterium glutamicum	21573			Bar bacterium	Corynebacterium glutamicum	21572
Bar bacterium	Corynebacterium glutamicum	21573			Bar bacterium	Corynebacterium glutamicum	21579
Bar bacterium	Corynebacterium glutamicum	19049			Bar bacterium	Corynebacterium glutamicum	21579
Bar bacterium	Corynebacterium glutamicum	19049			Bar bacterium	Corynebacterium glutamicum	19050
Bar bacterium	Corynebacterium glutamicum	19051			Bar bacterium	Corynebacterium glutamicum	19050
Bar bacterium	Corynebacterium glutamicum	19051			Bar bacterium	Corynebacterium glutamicum	19052
Bar bacterium	Corynebacterium glutamicum	19053			Bar bacterium	Corynebacterium glutamicum	19052
Bar bacterium	Corynebacterium glutamicum	19053			Bar bacterium	Corynebacterium glutamicum	19054
Bar bacterium	Corynebacterium glutamicum	19055			Bar bacterium	Corynebacterium glutamicum	19054
Bar bacterium	Corynebacterium glutamicum	19055			Bar bacterium	Corynebacterium glutamicum	19056
Bar bacterium	Corynebacterium glutamicum	19057			Bar bacterium	Corynebacterium glutamicum	19056
Bar bacterium	Corynebacterium glutamicum	19057			Bar bacterium	Corynebacterium glutamicum	19058
Bar bacterium	Corynebacterium glutamicum	19059			Bar bacterium	Corynebacterium glutamicum	19058
Bar bacterium	Corynebacterium glutamicum	19059			Bar bacterium	Corynebacterium glutamicum	19060
Bar bacterium	Corynebacterium glutamicum	19185			Bar bacterium	Corynebacterium glutamicum	19060
Bar bacterium	Corynebacterium glutamicum	19185			Bar bacterium	Corynebacterium glutamicum	13286
Bar bacterium	Corynebacterium glutamicum	21515			Bar bacterium	Corynebacterium glutamicum	13286
Bar bacterium	Corynebacterium glutamicum	21515			Bar bacterium	Corynebacterium glutamicum	21527
Bar bacterium	Corynebacterium glutamicum	21544			Bar bacterium	Corynebacterium glutamicum	21527
Bar bacterium	Corynebacterium glutamicum	21544			Bar bacterium	Corynebacterium glutamicum	21492
Bar bacterium	Corynebacterium glutamicum		B8183		Bar bacterium	Corynebacterium glutamicum	21492
Bar bacterium	Corynebacterium glutamicum		B8183		Bar bacterium	Corynebacterium glutamicum			B8182
Bar bacterium	Corynebacterium glutamicum		B12416		Bar bacterium	Corynebacterium glutamicum			B8182
Bar bacterium	Corynebacterium glutamicum		B12416		Bar bacterium	Corynebacterium glutamicum			B12417
Bar bacterium	Corynebacterium glutamicum		B12418		Bar bacterium	Corynebacterium glutamicum			B12417
Bar bacterium	Corynebacterium glutamicum		B12418		Bar bacterium	Corynebacterium glutamicum			B11476
Bar bacterium	Corynebacterium glutamicum	21608			Bar bacterium	Corynebacterium glutamicum			B11476
Bar bacterium	Corynebacterium glutamicum	21608			Bar bacterium	Lily bar bacterium		P973
Bar bacterium	nitrilophilus	21419		11594	Bar bacterium	Lily bar bacterium		P973
Bar bacterium	nitrilophilus	21419		11594	Bar bacterium	Kind		P4445

Bar bacterium	Kind		P4446
Bar bacterium	Kind		P4446		Bar bacterium	Kind	31088
Bar bacterium	Kind	31089			Bar bacterium	Kind	31088
Bar bacterium	Kind	31089			Bar bacterium	Kind	31090
Bar bacterium	Kind	31090			Bar bacterium	Kind	31090
Bar bacterium	Kind	31090			Bar bacterium	Kind	31090
Bar bacterium	Kind	15954		20145	Bar bacterium	Kind	31090
Bar bacterium	Kind	15954		20145	Bar bacterium	Kind	21857
Bar bacterium	Kind	21862			Bar bacterium	Kind	21857
Bar bacterium	Kind	21862			Bar bacterium	Kind	21863

ATCC: American type culture collection, Rockville, MD, USA

FERM: fermentation research institute, Chiba, Japan

NRRL: agricultural research institute's collection, northern areas research laboratory, Peoria, IL, USA

CECT: Spain's Type Tissue Collection, Valencia, Spain

NCIMB: state-run industry and Marine Bacteria center, Aberdeen, UK

CBS: fungi strain collection, Baarn, NL

NCTC: state-run Type Tissue Collection, London, UK

DSMZ: Mikroorganismen collection, Braunschweig, Germany

It can be found in Sugawara, H.et al. (1993) World directory of collections of cultures of microorganisms:Bacteria, fungi and yeasts (4^thEdn), World federation for culture collections world data center onmicroorganisms, Saimata, Japen.

Table 4: sequence comparison result

ID#	Genbank hit results (NT)	Length	Searching number	Genbank hit results title	Genbank hit results source	% homology (GAP)	It is put in storage day
ID#	Genbank hit results (NT)	Length	Searching number	Genbank hit results title	Genbank hit results source	% homology (GAP)	It is put in storage day	rxa00315	1527 GB_BA1:AB007125 GB_IN1:CELC47D2 GB_HTG2:AC006732	4078 17381 159453	AB007125 U64861 AC006732	Serratia marcescens slaA gene for surface layer protein, completecds, isolate 8000Caenorhabditis elegans cosmid C47D2.Caenorhabditis elegans clone Y32G9,^*SEQUENCING INPROGRESS^*, 9 unordered pieces.	Serratia marcescens Caenorhabditis elegans Caenorhabditis elegans	40,386 36,207 36,436	26-MAR-1998 28-Jul-96 23-Feb-99
rxa01503	372 GB_PR3:AC005019 GB_GSS12:AQ390040 GB_GSS5:AQ784231	188362 680 542	AC005019 AQ390040 AQ784231	Homo sapiens BAC clone GS250A16 from 7p21-p22, completeRPCI11-157C9.TJ RPCI-11 Homo sapiens genomic clone RPCI-11-157C9, genomic survey sequence.HS_3087_B1_C10_T7C CIT Approved Human Genomic SpermLibrary D Homo sapiens genomic clone Plate=3087 Col=19Row=F, ge Nomic survey sequence.	Homo sapiens Homo sapiens Homo sapiens	39,722 43,137 37,643	27-Aug-98 21-MAY-1999 3-Aug-99	rxa00315	1527 GB_BA1:AB007125 GB_IN1:CELC47D2 GB_HTG2:AC006732	4078 17381 159453	AB007125 U64861 AC006732			40,386 36,207 36,436	26-MAR-1998 28-Jul-96 23-Feb-99
rxa01503	372 GB_PR3:AC005019 GB_GSS12:AQ390040 GB_GSS5:AQ784231	188362 680 542	AC005019 AQ390040 AQ784231		Homo sapiens Homo sapiens Homo sapiens	39,722 43,137 37,643	27-Aug-98 21-MAY-1999 3-Aug-99	rxa01299	2187 GB_EST38:AW047296 GB_RO:AB004056 GB_RO:AB004056	614 1581 1581	AW047296 AB004056 AB004056	UI-M-BH1-amh-e-03-0-UI.s1 NIH_BMAP_M_S2 Mus musculuscDNA clone UI-M-BH1-amh-e-03-0-UI 3 ', mRNA sequenceRattus norvegicus mRNA for BarH-class homeodomaintranscription factor.complete cds.Rattus norvegicus mRNA for BarH-class homeodomaintranscription factor, c Omplete cds.	Mus musculus Rattus norvegicus Rattus norvegicus	41,475 41,031 40,717	18-Sep-99 2-Sep-98 2-Sep-98
rxa00951	416 GB_BA1:SCJ21 GB_VI:MCU68299 GB_VI:U93872	31717 230278 133661	AL109747 U68299 U93872	1 complete genomic sequence.Kaposi ' s sarcoma-associated herpesvirus glycoprotein M of Streptomyces coelicclor cosmid J21. Mouse cytomegalovirus, DNAreplication protein, glycoprotein, DNA replication protein, FLICEinhibitory protein and v-cyclin genes, complete cds , and tegument	Streptomyces coelicolor A3(2) Mouse cytomegalovirus 1 Kaposi′s sarcoma- associated herpesvirus	34,913 40,097 36,029	5-Aug-99 04-DEC-1996 9-Jul-97	rxa01299	2187 GB_EST38:AW047296 GB_RO:AB004056 GB_RO:AB004056	614 1581 1581	AW047296 AB004056 AB004056		Mus musculus Rattus norvegicus Rattus norvegicus	41,475 41,031 40,717	18-Sep-99 2-Sep-98 2-Sep-98
rxa00951	416 GB_BA1:SCJ21 GB_VI:MCU68299 GB_VI:U93872	31717 230278 133661	AL109747 U68299 U93872			34,913 40,097 36,029	5-Aug-99 04-DEC-1996 9-Jul-97	rxa01244	1827 GB_BA1:AFAPHBHI GB_PR3:HSJ836E13 GB_EST24:AI170227	4501 78055 409	M69036 AL050326 AI170227	Alcaligenes eutrophus protein H (phbH) and protein I (phbI) genes, complete cds.Human DNA sequence from clone 836E13 on chromosome 20Contains ESTs, STS and GSSs, complete sequence.EST216152 Normalized rat lung, Banto Soares Rattus sp.cDNAclone RLUCF56 3 ' end, mRNA s Equence.	Ralstonia eutropha Homo sapiens Rattus sp.	45,624 37,303 39,098	26-Apr-93 23-Nov-99 20-Jan-99
rxa01300	390 GB_PR3:HUMDODDA GB_PAT:140899 GB_PAT:140900	26764 26764 1317	L39874 I40899 I40900	1 from patent US 5622851.Sequence of Homo sapiens deoxycytidylate deaminase gene, complete cds.Sequence, 2 from patent US 5622851.	Homo sapiens Unknown. Unknown.	37,644 37,644 37,644	11-Aug-95 13-MAY-1997 13-MAY-1997	rxa01244	1827 GB_BA1:AFAPHBHI GB_PR3:HSJ836E13 GB_EST24:AI170227	4501 78055 409	M69036 AL050326 AI170227		Ralstonia eutropha Homo sapiens Rattus sp.	45,624 37,303 39,098	26-Apr-93 23-Nov-99 20-Jan-99
rxa01300	390 GB_PR3:HUMDODDA GB_PAT:140899 GB_PAT:140900	26764 26764 1317	L39874 I40899 I40900		Homo sapiens Unknown. Unknown.	37,644 37,644 37,644	11-Aug-95 13-MAY-1997 13-MAY-1997	rxa00953	789 GB_BA1:SCJ21 GB_BA1:BLTRP GB_PAT:E01375	31717 7725 7726	AL 109747 X04960 E01375	Streptomyces coelicolor cosmid J21. Brevibacterium lactofermentum tryptophan operon. DNA sequence of tryptophan operon	Streptomyces coelicolor A3(2) Corynebacterium glutamicum Corynebacterium glutamicum	39,398 39,610 46,753	5-Aug-99 10-Feb-99 29-Sep-97

4 (Continued) of table

rxa01943

2172 GB_BA1:CORPTSMA GB_BA1:BRLPTSG GB_BA2:AF045481

2656 3163 2841

L18874 L18875 AF045481

Corynebacterium glutamicum phosphoenolpyruvate sugar phosphotransferase (ptsM) mRNA, complete cds. Brevibacterium lactofermentum phosphoenolpyruvate sugar phosphotransferase (ptsG) gene.complete cds. Corynebacterium ammoniagenes glucose permease (ptsG) gene , complete cds

Corynebacterium glutamicum Brevibacterium lactofermentum Corynebacterium ammoniagenes

100,000 84,963 53,558

24-Nov-94 01-OCT-1993 29-Jul-98

Embodiment

Embodiment 1: the preparation of 13032 full gene group DNA of Corynebacterium glutamicum ATCC

Corynebacterium glutamicum (ATCC 13032) culture in BHI culture medium (Difco), stay overnight by 30 DEG C of violent shaken cultivations.Cell is collected by centrifugation, abandons supernatant, cell is resuspended in 5ml buffer I (all volumes pointed out of the 5%- of culture original volume are calculated for 100ml culture volume).The composition of buffer I: 140.34g/l sucrose, 2.46g/l MgSO₄×7H₂O, 10ml/l KH₂PO₄Solution (100g/l, KOH are adjusted to PH6.7), 50g/l M12 concentrate (10g/l (NH₄)₂SO₄, 1g/l NaCl, 2g/l MgSO₄× 7H2O, 0.2g/l CaCl₂, 0.5g/l yeast extract (Difco)), 10ml/l trace element mixture (200mg/l FeSO₄×H₂O, 10mg/l ZnSO₄×7H₂O, 3mg/l MnCl₂×4H₂O, 30mg/l H₃BO₃, 20mg/lCoCl₂×6H₂O, 1mg/l NiCl₂×6H₂O, 3mg/l Na₂MoO₄×2H₂O), 500mg/l complexing agent (EDTA or citric acid), 100ml/l vitamin mixtures (0.2mg/l biotin, 0.2mg/l folic acid, 20mg/l p- amino benzoic acid, 20mg/l riboflavin, 40mg/lpanthothenate, 140mg/l niacin, 40mg/l hydrochloric acid Vitamin B6,200mg/l inositol).Lysozyme is added in suspension to final concentration 2.5mg/ml.After 37 DEG C are incubated for about 4 hours, cell wall is degraded, and obtained protoplast is with being collected by centrifugation.Precipitating is washed once with 5ml buffer I, is washed once with 5ml TE buffer (10mM Tris-HCl, 1ml EDTA, pH8).Precipitating is resuspended with 4ml TE buffer, and 0.5ml SDS solution (10%) and 0.5ml NaCl solution (5M) is added.Proteinase K is added to 200 μ g/ml of final concentration, suspension is incubated for about 18 hours at 37 DEG C.DNA phenol, phenol chloroform-isoamyl alcohol, chloroform-isoamyl alcohol are according to standardization program extraction purification.Then, the 3M sodium acetate of 1/50 volume and the ethyl alcohol of 2 times of volumes is added, is incubated for 30 minutes at -20 DEG C, with the supercentrifuge 12 for using SS34 rotary head (Sorvall), 000rpm is centrifuged 30 minutes, precipitates DNA.DNA is dissolved in the 1ml TE buffer containing 20 μ g/ml RNaseA, is dialysed at least 3 hours for 4 DEG C in 1000ml TE buffer.In this period, replace buffer 3 times.In the DNA solution of every 0.4ml dialysis, 0.4ml 2M LiCl and 0.8ml ethyl alcohol is added.After -20 DEG C are incubated for 30 minutes, DNA is collected in centrifugation (13,000rpm, Biofuge Fresco, Heraeus, Hanau, Germany).DNA precipitating is melted in TE buffer.It can be used for all purposes by DNA prepared by the program, the building including southern hybridization and genomic library.

Embodiment 2: in the building of the genomic library of Escherichia coli Glutamic Acid bar bacterium ATCC13032

Use the DNA prepared as described in Example 1, according to method that is known and sufficiently establishing (referring to, such as Sambrook, J.et al. (1989) " Molecular Cloning:ALaboratory Manual " Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, or Ausubel, F.M.et al. (1994) " Current Protocols inMolecular Bilogy ", John Wiley&Sons.), can construct cosmid library and Plasmid library.

Any plasmid and clay can be used.PBR322 plasmid (Sutcliffe, J.G. (1979) Proc.Natl.Acad.Sci.USA, 75:3737-3741)；PACY177 (Change&Cohen (1978) J.Bacteriol 134:1141-1156), pBS series plasmids (pBSSK+, pBSSK- and other plasmids；Stratagene, LaJolla, USA), clay SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A.and Waterson, R.H. (1987) Gene53:283-286) it can be used for specific use.The gene library specially used in corynebacterium glutamicum can be constructed with plasmid pSL109 (Lee, H.-S.and A.J.Sinskey (1994) J.Microbiol.Biotechnol.4:256-263).

Embodiment 3:DNA sequencing and computer function analysis

According to standard method, use such as described genomic library in example 2, it can carry out DNA sequencing, especially with using ABI377 sequenator chain termination method (referring to, such as Fleischman, R.D.et al. (1995) " Whole-genome Random Sequencing andAssembly of Haemophilus Influenzae Rd., Science, 269:496-512).Use the sequencing primer with following nucleotide sequence: 5 '-GGAAACAGTATGACCATG-3 ' or 5 '-GTAAAACGACGGCCAGT-3 '.

Embodiment 4: vivo mutations

Can by Escherichia coli or other microorganisms (such as, the certain bacterium of bacillus or seem saccharomyces cerevisiae yeast) plasmid (or other carriers) DNA passage, the vivo mutations of Corynebacterium glutamicum are carried out, wherein these microorganisms keep the ability of its hereditary information globality to be damaged.Typical mutant strain has mutation (for example, mutHLS, mutD, mutT etc. in the gene of DNA repair system；Bibliography is referring to Rupp, W.D. (1996) DNA repair mechanisms, in:Escherichia coli and Salmonella, p.2277-2294, ASM:Washington.).These bacterial strains are well known for the man skilled in the art.The use of these bacterial strains is set forth in, such as Greener, in A.and Callahan, M. (1994) Strategies 7:32-34.

Embodiment 5: the DNA transmitted between Escherichia coli and Corynebacterium glutamicum

Bar bacterium and brevibacterium strain contain the indigenous plasmid (seeming for example, pHM1519 or pBL1) of the spontaneous duplication of energy (comment is see, e.g., Martin, J.F.et al. (1987) Biotechnology, 5:137-146).The shuttle vector of Escherichia coli and Corynebacterium glutamicum, the standard vector that Escherichia coli can be used readily constructs (Sambrook, J.et al. (1989) " Molecular Cloning:A Laboratory Manual " Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press or Ausubel, F.M.et al. (1994) " Current Protocols in Molecular Bilogy ", John Wiley&Sons.), i.e., glutamic acid is added wherein The replication fork starting point of bar bacterium and suitable label.This replication origin, what the indigenous plasmid preferably separated from bar bacterium and brevibacterium strain obtained.That as these strain transformation markers this specific uses is kalamycin resistance gene (such as from those of Tn5 Tn903 transposons kalamycin resistance gene) or chloramphenicol resistance gene (Winnacker, E.L. (1987) " From Genes to Clones-Introduction to Gene Technology; VCH, Weinheim).Constructing many of the document of various wild type shuttle vectors example, these shuttle vectors can replicate in Escherichia coli and Corynebacterium glutamicum, and it can be used for various purposes, including gene overexpression, (bibliography is see, e.g., Yoshihama, M.et al. (1985) J.Bacteriol.162:591-597, Martin J.F.et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B.J.et a；(1991) Gene, 102:93-98).

Can be interested gene cloning into above-mentioned shuttle vector using standard method, and the hybridization can be carried and be introduced into Corynebacterium glutamicum strain.The conversion of Corynebacterium glutamicum can pass through protoplast transformation (Kastsumata, R.et al. (1984) J.Bacteriol.159306-311), fax hole (Liebl, E.et al. (1989) FEMS Microbiol.Letters, 53:399-303) realize, when using special carrier, it can also be realized by combination (such as in Sch fer, A et al. (1990) J.Bacteriol.172:1663-1666).Shuttle vector can also be transferred to Escherichia coli from Corynebacterium glutamicum by preparing Plasmid DNA (using standard method known in the art) from Corynebacterium glutamicum and being transformed into Escherichia coli.Standard method progress can be used in this step of converting, but Mcr defective escherichia coli bacterial strain, such as NM522 (Gough&Murray (1983) J.Mol.Biol.166:1-19) is used to be advantageous.

Using contain pCG1 (U.S.Patent No.4,617, or the plasmid of its segment 267), and it can choose the kalamycin resistance gene (Grindley from TN903, N.D.and Joyce, C.M. (1980) Proc.Natl.Acad.Sci.USA 77 (12): 7176-7180), so that it may overexpression gene in corynebacterium glutamicum.In addition, can also overexpression gene in corynebacterium glutamicum using plasmid pSL109 (Lee, H.-S.and A.J.Sinskey (1994) J.Microbiol.Biotechnol.4:256-263).

Other than using reproducible plasmid, the overexpression of gene can also be realized by genome conformity.The genome conformity of Corynebacterium glutamicum or other bar bacteriums or brevibacterium strain, it can be realized by well known method, such as the homologous recombination of genome area, restriction endonuclease mediate integration (REMI) (see, for example, DE Patent 19823834), or by using transposons.Can also by modification adjustment region (such as, promoter, repressor and/or enhancer), it is based on sequence modification, insertion or the missing of chance event method (such as transposon mutant or REMI), either by using site-directed method (such as homologous recombination) to adjust the activity of gene of interest.Nucleic acid sequence as transcription terminator can also be inserted into the 3 ' of one or more gene coding region of the invention；What such terminator was well known, and describe in such as Winnacker, E.L. (1987) From Genes to Clones-Introduction to Gene Technology.VCH:Weinheim.

Embodiment 6: the estimation of mutein expression

The fact that be converted the active observation of mutein in host cell, depend on, i.e. mutein are expressed in a manner of similar with wild-type protein with similar quantity.Determine mutated gene transcriptional level (quantitative index of the mRNA for gene product translation) the useful method of one kind be carry out Northern hybridization (bibliography referring to, such as, Ausubel et al. (1988) CurrentProtocols in Molecular Biology, Wiley:New York), that wherein designs has detectable label (usually radioactive or chemiluminescent) for the primer mark in conjunction with gene of interest, to, when whole RNA of organism culture are extracted, run gel electrophoresis, it is transferred in stable and is incubated for the probe, the combination of bonding probes and quantity just indicate the presence and quantity of the gene mRNA.The information is the evidence of mutated gene transcription degree.Several method can be used and prepare whole cell RNAs from Corynebacterium glutamicum, what this was well known, such as description is in Bormann, E.R.et al. (1992) Mol.Microbiol.6:317-326.

In order to estimate by the presence and relative populations of the mRNA protein translated, it can be used standard technique, such as Wesstern hybridization (see, e.g., Ausubel et al. (1988) CurrentProtocols in Molecular Biology, Wiley:New York).In the method, whole cell proteins are extracted, are separated by gel electrophoresis, is transferred to and seems on medium as nitrocellulose, and are incubated for altogether with the probe of required protein specific bond, such as antibody.The probe usually marks chemiluminescent or colorimetric the label for having detection.The presence and quantity of the label observed indicate the presence and quantity for appearing in the required mutein in cell.

Embodiment 7: the growth-medium and condition of culture of the Corynebacterium glutamicum of genetic modification

The Corynebacterium glutamicum of genetic modification can be cultivated in synthesis or spontaneous growth culture medium.A variety of different growth mediums for Corynebacterium glutamicum are known and are readily available (Lieb, et al. (1989) Appl.Microbiol.Biotechno., 32:205-210；Von der Ostenet al. (1998) Biotechnology Letters, 11:11-16；Patent DE 4,120,867；Liebl (1992) " The Genus Corynebacterium, in:Procaryotes, Volume II, Balows, A.et al., eds.Springer-Verlag).These culture mediums contain one or more kinds of carbon sources, nitrogen source, inorganic salts, vitamin and microelement.Preferred carbon source is carbohydrate, such as monosaccharide, disaccharides or polysaccharide.For example, glucose, fructose, mannose, galactolipin, ribose, sorbose, ribulose, lactose, maltose, sucrose, gossypose, starch or cellulose, are used as good carbon source.Carbohydrate, such as molasses or the by-product of other carbohydrates refining can also be provided to culture medium by complex compound.The mixture for improving different carbon source is also advantageous.Other available carbon sources have alcohol and organic acid, such as methanol, ethyl alcohol, acetic acid or lactic acid.Nitrogen source is usually organic perhaps inorganic nitrogen compound or the substance containing these compounds.Representative nitrogen source includes ammonia or ammonium salt, such as NH₄Cl or (NH₄)₂SO₄、NH₄OH, nitrate, urea, amino acid perhaps complexity nitrogen source for example corn steep liquor, soy meal, soybean protein, yeast extract, meat extract or other.

It may include inorganic salt compound in the medium, calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper or iron including hydrochloride, phosphate or sulfate.Chelating agent may be added in culture medium, to maintain the metal ion in solution.Particularly useful chelating agent includes dihydroxy phenol, seems catechol and protocatechuic acid or organic acid, seems citric acid.Culture medium typically also contains growth factor, such as vitamin and growth promoter, their example includes biotin, riboflavin, thiamines, folic acid, niacin, pantothenate and pyridoxol.Growth factor and salt are often from complicated medium component, such as yeast extract, molasses, corn steep liquor and other compositions.The definite composition of culture based compound is strongly dependent upon direct experiment, and for each concrete condition concrete decision.The information optimized about culture medium passes through in textbook " AppliedMicrobiol.Physiology; A Practical Approach " (eds.P.M.Rhodes, P.F.Stanbury, IRL Press (1997) pp.53-73, ISBN 0 19 is 9635773) " in.It can also seem standard 1 (Merck) perhaps BHI (grain heart infusion DIFCO) or others from commercial supplier there selection growth medium.

All nutrient media components will pass through heating (1.5bar, 120 DEG C, 20 minutes) or the sterilizing that is sterile filtered.Component can sterilize together, or if necessary separate individually sterilizing.All nutrient media components can just be added in the beginning of growth, either can choose continuity or be added portionwise.

Condition of culture determines each experiment respectively.Temperature should be within the scope of 15 DEG C to 45 DEG C.Temperature can be kept constant, or be changed in an experiment.The pH of culture medium is in 5 to 8.5 ranges, preferably about 7.0, and can be maintained by the addition of buffer in culture medium.It is kaliumphosphate buffer for the representational buffer of this purpose.Buffer, such as MOPS, HEPES, ACES and others are synthesized, also can replace use or is used simultaneously.Addition NaOH or NH can also be passed through during the growth process₄OH, to maintain stable culture pH.If using seeming complexity nutrient media components as yeast extract, it is possible to reduce the necessity of buffer is added, this is because many complex compounds have the fact that very strong buffer capacity.If ammonia control pH also can be used using fermentation tank culture microorganism.

Incubation time is usually within a few houres to several days.The selection of this time is to allow to accumulate the maximum amount of product in liquid medium.The growth experiment of announcement can be to carry out in various containers, for example, microtiter plate, teat glass, glass shaking flask or different size of glass or metal fermentor.In order to screen a large amount of clone, microorganism should be cultivated in having baffle or the not microtiter plate of baffle, teat glass or shaking flask.100ml shaking flask is preferably used, the required culture medium of 10% (volume) is added.Shaking flask should be placed on shake (25 millimeters of amplitude), velocity interval 100-300rpm on shaking table.It can be by keeping wet air to reduce evaporation loss；Alternatively, carrying out mathematics amendment to evaporation loss.

If detecting the clone of genetic modification, it should also detect unmodified control clone or be cloned containing basic plasmid but without the control of any insertion.Use growth 30 DEG C of cells being incubated for, such as CM plate (10g/l glucose, 2.5g/l NaCl, 2g/l urea on a lbmc agar plate, the more peptones of 10g/l, 5g/l yeast extract, 5g/l gravy extract, 22g/l agar, 2M NaOH are adjusted to pH 6.8), inoculation medium to OD₆₀₀Value is 0.5-1.5.The inoculation of culture medium can be realized by the salt suspensioning liquid of Corynebacterium glutamicum cell of the introducing from CM plate, or the liquid pre-culture by the way that the bacterium is added.

Embodiment 8: the analyzed in vitro of mutein function

The measurement of the activity and kinetic parameter of enzyme has technically established very well.It is any that the given determination of activity by the enzyme changed is tested, it is necessary to be suitble to the sp act of wild-type enzyme, this is completely within the ability of technology skilful person.It, can be for example below with reference to being found in document: Dixon about the Summarizing comment of enzyme, and about structure, dynamics, principle, method, application and the clear details for determining many enzymatic activity examples, M., and Webb, E.C., (1979) Enzymes.Longmans:London；Fersht, (1985) Enzyme Structure and Mechanism.Freeman:NewYork；Walsh, (1979) Enzymatic Reaction Mechanisms.Freeman:San Francisco；Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology.Oxford Univ.Press:Oxford；Boyer, P.D., ed. (1983) The Enzymes, 3^rdEd.Academic Press:New York；Bisswanger, H., (1994) Enzymkinetik, 2^ndEd.VCH:Weinheim (ISBN 3527300325)；Bergmeyer, H.U., Bergmeyer, J., Gra β l, M., eds. (1983-1986) Methods of EnzymaticAnalysis, 3^rdEd., vol.I-XII, Verlag Chemie:Weinheim；And Ullmann ' s Encyclopediaof Industrial Chemistry (1987) vol.A9, " Enzymes " .VCH:Weinheim, p.352-363.

It can be measured by several methods known in the art in conjunction with the activity of the protein of DNA, such as DNA band shift analysis (also referred to as gel retardation assay).Effect of these protein to other developed by molecule, can be measured with reporter gene assays (such as description is in Kolmar, H.et al. (1995) EMBO J.14:3895-3904 in, and its bibliography of reference).Reporter gene test systems are known, and the application in protokaryon and eukaryocyte all has been established, using seeming BgaA, enzyme as green fluorescent protein and other several protein.

The active measurement of protein called membrane transporters matter can be according to such as description in Gennis, R.B. (1989) " Pores, Channels and Transporters ", in Biomembrane, Molecular Structureand Function, Springer:Heidelberg, p.85-137；199-234；Technology carries out those of in and 270-322.

Embodiment 9: the analysis for the effect that mutein produces required product

The effect that Corynebacterium glutamicum genetic modification produces required compound (such as amino acid), it can estimate in this way, the microorganism modified is grown by (such as those described above) under appropraite condition, and analyze the culture medium and/or cellular component for increasing required product (for example, amino acid) production.These analytical technologies are well known for skilled ordinary skill, including spectrum analysis, thin-layer chromatography, various colouring methods, enzymatic method and microbial process, it and seem high performance liquid chromatography (Ullman, Encyclopedia of Industrial Chemistry, vol.A2, p.89-90 andp.443-613, VCH:Weinheim (1985)；Fallon, A.et al., (1987) " Applications ofHPLC in Biochemistry " in:Laboratory Techniques in Biochemistry and MolecularBiology, vol.17；Rehm et al. (1993) Biotechnology, vol.3, Chapter III: " Productrecovery and purification ", page 469-714, VCH:Weinheim；Belter, P.A.et al. (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) Biochemicalseparations, in:Ulmann ' s Encyclopedia of Industrial Chemistry, vol.B3, Chapter11, page 1-27, VCH:Weinheim；And Dechow, F.J. (1989) Separation and purificationtechniques in biotechnology, Noyes Publications) such analysis chromatography.

In addition to the measurement to final tunning, the other components of the metabolic pathway for required production of chemicals can also be analyzed, such as intermediate and by-product, to determine the production capacity of organism, yield, and/or the production efficiency of compound.Analysis method include nutrient level in culture medium (such as, carbohydrate, hydrocarbon, nitrogen source, phosphoric acid and other ions) measurement, the measurement of biomass composition and growth, the analysis of the production of biosynthesis pathway common metabolic product, and the measurement to gas is generated in fermentation.In Applied MicrobialPhysiology, A Practical Approach, P.M.Rhodes and P.F.Stanbury, eds., IRL Press, p.103-163 the standard method of these measurements outlines；And 165-192 (ISBN:0199635773) and its reference bibliography in.

Embodiment 10: the purifying of required product in Corynebacterium glutamicum culture

Required product is recycled from Corynebacterium glutamicum cell or in the supernatant of above-mentioned culture medium, can be carried out by various methods known in the art.If required product is not that cell is secreted, cell can be collected from culture medium by low-speed centrifugal, with standard technique lytic cell, such as mechanical force or ultrasonic wave.It is centrifuged off cell fragment, retains the supernatant fraction containing soluble protein for required compound to be further purified.If product is secreted from Corynebacterium glutamicum cell, cell is removed from culture medium with low-speed centrifugal, retains supernatant fraction for being further purified.

The supernatant fraction that any purification process obtains is chromatographed with suitable resin, and required molecule is retained by chromatographic resin, and many impurity in sample are not preserved or impurity is retained by resin, and sample is not preserved.Using same or different chromatographic resin, this chromatographic step of repetition can according to need.It is familiar with the suitable chromatographic resin of the selection that ordinary skill can be very skilled, and knows the application most effective for specific molecular to be purified of these resins.The product of purifying can use filtering or ultrafiltration concentration, and be stored at the maximum temperature of product stability.

Purification process known in the art is very more, and purification process above-mentioned is not meant to be limited only to this.The description of these purification process exists, such as in Bailey, J.E.& Ollis, D.F.BiochmicalEngineering Fundamentals, McGraw-Hill:New York (1986).

The characteristic and purity for separating compound, can be estimated with technical standard technique.This includes high performance liquid chromatography (HPLC), light-splitting method, colouring method, thin-layer chromatography, NIRS, enzymatic method or microbial process.These analysis methods have comment in the following documents: Patek et al. (1994) Appl.Environ.Microbiol.60:133-140；Malakhova et al. (1996) Biotekhnologiya 11:27-32；And Schmidt et al. (1998) Bioprocess Engineer.19:67-70.Ulmann ' sEncyclopedia of Industrial Chemistry, (1996) vol.A27, VCH:Weinheim, p.89-90, p.521-540, p.540-547, p.559-566,575-581 and is p.581-587；Michal, G. (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, JohnWiley and Sons；Fallon, A.et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, vol.17.

Embodiment 11: the analysis of gene order of the present invention

Sequence compares the measurement of the percent homology between two sequences, it is technology known in the art, the completion of mathematical operation rule can be used, such as the algorithm in Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87:2264-68, the algorithm have modification in Karlin andAltschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-77.The algorithm is incorporated into Altschul, in NBLAST the and XBLAST program (2.0 editions) in et al. (1990) J.Mol.Biol.215:403-10.BLAST nucleotide searches can be carried out with NBLAST program, score=100, wordlength=12, the available nucleotide sequence with PTS nucleic acid molecule homologous of the present invention.BLAST protein searches can be carried out with XBLAST program, score=50, wordlength=3, the available amino acid sequence with pts protein matter molecule homologous of the present invention.For comparison purposes, in order to obtain the alignment in gap, description can be used in Altschul et al., (1997) Nucleic Acids Res.25 (17): the GappedBLAST in 3389-3402.When using BLAST and Gapped blast program, it is familiar with ordinary skill knows the parameter for how optimizing program (for example, XBLAST and NBLAST) for specific sequence to be analyzed.

Another mathematical operation rule example compared for sequence is Meyers and Miller algorithm ((1998) Comput.Appl.Biosci.4:11-17).The algorithm is incorporated into ALIGN program (2.0 editions), which is a part of GCG sequence nucleotide sequence comparison software packet.When using ALIGN program comparing amino acid sequence, PAM120 weight residue table, gap length punishment 12, gap punishment 4 can be used.Other sequence analytic operation rules are technically also known, including ADVANCE and ADAM, are described in Torelli and Robotti (1994) Comput.Appl.Biosci.10:3-5；And FASTA, it describes in Pearson and Lipman (1998) P.N.A.S.85:2444-8.

The realization of the GAP program in GCG software package (http://www.gcg.com is provided with) also can be used in Percent homology between two amino acid sequences, use 250 matrix of 62 matrix of Blosum or PAM, the perhaps 4 length component 2,3 or 4 of gap component 12,10,8,6.The realization of the GAP program in GCG software package can be used in Percent homology between two nucleic acid sequences, uses standard parameter, such as gap component 50 and length component 3.

Comparative analysis in gene order of the present invention and Genbank between sequence, can be used technology known in the art carry out (referring to, such as, Bexevanis and Ouellette, eds. (1998) Bioinformatics:A Practical Guide to the Analysis of Genes and Proteins.John Wiley and Sons:New York).Gene order of the present invention is compared by the method for three steps with the sequence in Genbank.In the first step, BLASTN analysis (for example, local sequence comparative analysis) is carried out with respect to the nucleotide sequence in Genbank to each sequence of the invention, retains highest 500 matchings and is further analysed.Then FASTA is made to this 500 matchings and searches (for example, local and global composite sequence comparative analysis, is wherein carrying out alignment to the sequence area of restriction).Next, matching to every gene order of the invention with three highests of FASTA, whole world alignment is carried out using the GAP program (using standard parameter) in GCG software package.Correct result in order to obtain, the sequence length selected from Genbank are adjusted to the length of search sequence using technical well known method.The result of the analysis is listed in Table 4.Although the result obtained in this way, obtained result is analyzed with the independent GAP (whole world) carried out is compareed relative to Genbank every to every gene of the invention, it is consistent, but for the GAP of large database concept (whole world) analysis, the required calculating time greatly reduces.The sequence of the present invention for not obtaining the above alignment of cutoff value shows to lack alignment's information in table 4.Being familiar with ordinary skill further can understand, GAP alignment's percent homology under the title " %homology (GPA) " listed in table 4, is to be listed with Digital European format, wherein ', ' represents decimal system point.For example, " 40,345 " represent " 40.345% " to the value in the column.

The building and operation of embodiment 12:DNA microarray

Sequence of the invention can be also used for DNA microarray and (known to the design of DNA array, methods and applications are technically, describe for example, Schena, M.te al. (1995) Science 270:467-470；Wodicka, L.et al. (1997) Nature Biotechnology 15:1359-1367；DeSaizieu, A.et al. (1998) Nature Biotechnology 16:45-48；And DeRisi, J.L.et al. (1997) Science 278:680-686) building and application.

Using solid, perhaps flexible support includes nitrocellulose, nylon, glass, silicon or other materials to DNA microarray.Nucleic acid molecules can be connected to surface in an orderly way.After suitable marker, other nucleic acid or mixtures of nucleic acids can be with fixed making nucleic acid molecular hybridizations, and marking can be used for the individual signal strength that monitoring and measurement determines region hybrid molecule.The opposite or absolute quantity for the whole or selected nucleic acid in nucleic acid samples or mixture that this method allows simultaneous quantitative applicable.Therefore, DNA microarray allows the analysis of a variety of (up to 6800 or more) similar expression of nucleic acid (see, for example, Schena, M. (1996) BioEssays 18 (5): 427-431).

Sequence of the present invention can be used for designing oligonucleotide primer, these primers can expand the determination region of one or more of Corynebacterium glutamicum genes by the nucleic acid amplification reaction as polymerase chain reaction.The selection and design of 5 ' or 3 ' oligonucleotide primers or suitable linker, the surface that the PCR product allowed is covalently attached to above-mentioned supporting dielectric (is also described in, for example, Schena, M.et al. (1995) Science 270:467-470).

Nucleic acid microarray can also be by such as in Wodicka, the synthesis building of original position oligonucleotide described in L.et al. (1997) NatureBiotechnology 15:1359-1367.By photolithography method, region exactly determined in matrix can be exposed in light.Blocking group is photo-labile, to be activated and add through subject nucleotide, but is covered up and can not see the region of light without any modification.Next protection and photoactivation circulation, allow in the synthesis for determining position difference oligonucleotide.Present invention determine that zonule can be synthesized on the micro-array by solid phase oligonucleotide.

The nucleic acid molecules of the present invention in sample or mixture of ribonucleotides are appeared in, it can be with microarray hybridization.These nucleic acid molecules can be marked according to standard method.Briefly, nucleic acid molecules (for example, mRNA molecule or DNA molecular) can be labeled for example, in reverse transcription or DNA synthesis and in conjunction with the isotope perhaps nucleotide of fluorescent marker.Labeling nucleic acid was described (such as in Schena, M.et al. (1995) supra with hybridizing for microarray；Wodicka, L.et al. (1997), supra；In and DeSaizieu A.et al. (1998), supra).The detection of hybrid molecule and to be quantitatively suitble to specific binding marker.Radioactive label can be detected, for example, described in M.at al. (1995) supra, fluorescent marker can also be detected, such as use the method for Shalon etal. (1996) Gemone Research 6:639-645 in Schena.

As described above, application of the sequence of the present invention in DNA microarray, allows the comparative analysis of different Corynebacterium glutamicum strain or other bar bacteriums.For example, the research changed in the bacterial strains based on individual transcription distribution diagrams can be promoted by nucleic acid array methodologies, and promote to it is specific and/or required seem the important gene of strain characteristics as pathogenic, production capacity and pressure tolerance identification.Equally, using nucleic acid array technology, the distribution diagram of gene expression of the present invention during fermentation reaction can also be compared.

Embodiment 13: the analysis (proteomics) of cell protein group dynamics

Gene, composition and method of the invention can be used for studying the interaction and dynamics of protein population, referred to as " proteomics ".Interested protein population includes, but it is not limited to, Corynebacterium glutamicum all protein group (such as, it compares up with the protein population of other organisms), under particular surroundings or metabolic conditions (such as, medium and high temperature of fermenting perhaps low temperature or high pH or low pH) active those protein or in those active protein of particular growth or stage of development.

Protein population, such as gel electrophoresis can be analyzed with various well known technologies.Cell protein can also can be used various electrophoretic techniques and be separated from each other for example, by cracking or extracting acquisition.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein isolate matter, is based largely on their molecular wt.Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) passes through equal point (this not only reflects amino acid sequence, but also has shown the posttranslational modification of protein) protein isolate matter.Another more preferred protein analysis method is that the continuous combination of IEF-PAGE and SDS-PAGE, referred to as 2-D- gel electrophoresis are (in such as Hermann et al. (1998) Electrophoresis 19:3217-3221；Fountoulakis et al. (1998) Electrophoresis 19:1193-1202；Langen et al. (1997) Electrophoresis 18:1184-1192；It is described in Antelmann et al. (1997) Electrophoresis18:1451-1463).

It can be shown by standard technique with the protein that these methods separate, such as by dyeing or marking.Suitably dyeing is technically known, including Coomassie brilliant blue, silver staining or fluorescent dye, such as Sypro Ruby (Molecular Probes).Include in Corynebacterium glutamicum culture medium radiolabeled amino acid or other protein precursors (for example,³⁵S- methionine,³⁵S- cysteine,¹⁴C- labeled amino acid,¹⁵N- amino acid,¹⁵NO₃Or¹⁵NH₄ ⁺Or¹³C- labeled amino acid), it can make these cells before its Separation of Proteins with regard to labelled protein.Similar, fluorescent marker also can be used.These labelled proteins can be extracted, are isolated and separated according to aforementioned techniques.

The protein shown with these technologies can be further analysed by measuring dyestuff or label used.Such as optical means can be used in the quantity of specific protein, is quantitatively determined, and can be compared with the quantity of other protein on same gel or on other gels.Can compare for example, by optics, spectrum analysis, gel images analysis and scanning, either by using photographic film or display, the protein on gel is compared.What these technologies were well known.

In order to determine the characteristic of specific protein, direct sequence measurement or other standards technology can be used.Such as, the sequencing of N- and/or C- end amino acid (such as Edman degradation) can be used, and mass spectral analysis (especially MALDI ESI technology (see, for example, Langen et al. (1997) Electrophoresis 18:1184-1192)).Protein sequence provided herein may be used as the Corynebacterium glutamicum protein identification carried out by these technologies.

The information obtained by these technologies, it can be used for the various modes of the different sample rooms modifications (for example, different organisms, fermentation time point, culture medium condition or biotic environment in other conditions) under comparison protein presence, activity, different biotic factors.The data that these tests obtain, it can be individual, or what is combined with other technologies is used for various applications, such as compare under specific condition the behavior of (such as metabolic condition) various organisms, increase the production capacity of the bacterial strain of production fine chemical, or increases the efficiency of fine chemical production.

Equivalents

Being familiar with ordinary skill will recognize, or can determine and routine experiment is only used only, and specific embodiment of the invention described herein has many equivalents.Following claim is intended to encompass these equivalents.

Sequence table

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tac cag ggc acc gtg ctt cct gtg ctg gtg gtt tct tgg att ctg gca 259

Tyr Gln Gly Thr Val Leu Pro Val Leu Val Val Ser Trp Ile Leu Ala

40 45 50

acg atc gag aag ttc ctg cac aag cga ctc aag ggc act gca gac ttc 307

Thr Ile Glu Lys Phe Leu His Lys Arg Leu Lys Gly Thr Ala Asp Phe

55 60 65

ctg atc act cca gtg ctg acg ttg ctg ctc acc gga ttc ctt aca ttc 355

Leu Ile Thr Pro Val Leu Thr Leu Leu Leu Thr Gly Phe Leu Thr Phe

70 75 80 85

atc gcc att ggc cca gca atg cgc tgg gtg ggc gat gtg ctg gca cac 403

Ile Ala Ile Gly Pro Ala Met Arg Trp Val Gly Asp Val Leu Ala His

90 95 100

ggt cta cag gga ctt tat gat ttc ggt ggt cca gtc ggc ggt ctg ctc 451

Gly Leu Gln Gly Leu Tyr Asp Phe Gly Gly Pro Val Gly Gly Leu Leu

105 110 115

ttc ggt ctg gtc tac tca cca atc gtc atc act ggt ctg cac cag tcc 499

Phe Gly Leu Val Tyr Ser Pro Ile Val Ile Thr Gly Leu His Gln Ser

120 125 130

ttc ccg cca att gag ctg gag ctg ttt aac cag ggt gga tcc ttc atc 547

Phe Pro Pro Ile Glu Leu Glu Leu Phe Asn Gln Gly Gly Ser Phe Ile

135 140 145

ttc gca acg gca tct atg gct aat atc gcc cag ggt gcg gca tgt ttg 595

Phe Ala Thr Ala Ser Met Ala Asn Ile Ala Gln Gly Ala Ala Cys Leu

150 155 160 165

gca gtg ttc ttc ctg gcg aag agt gaa aag ctc aag ggc ctt gca ggt 643

Ala Val Phe Phe Leu Ala Lys Ser Glu Lys Leu Lys Gly Leu Ala Gly

170 175 180

gct tca ggt gtc tcc gct gtt ctt ggt att acg gag cct gcg atc ttc 691

Ala Ser Gly Val Ser Ala Val Leu Gly Ile Thr Glu Pro Ala Ile Phe

185 190 195

ggt gtg aac ctt cgc ctg cgc tgg ccg ttc ttc atc ggt atc ggt acc 739

Gly Val Asn Leu Arg Leu Arg Trp Pro Phe Phe Ile Gly Ile Gly Thr

200 205 210

gca gct atc ggt ggc gct ttg att gca ctc ttt aat atc aag gca gtt 787

Ala Ala Ile Gly Gly Ala Leu Ile Ala Leu Phe Asn Ile Lys Ala Val

215 220 225

gcg ttg ggc gct gca ggt ttc ttg ggt gtt gtt tct att gat gct cca 835

Ala Leu Gly Ala Ala Gly Phe Leu Gly Val Val Ser Ile Asp Ala Pro

230 235 240 245

gat atg gtc atg ttc ttg gtg tgt gca gtt gtt acc ttc ttc atc gca 883

Asp Met Val Met Phe Leu Val Cys Ala Val Val Thr Phe Phe Ile Ala

250 255 260

ttc ggc gca gcg att gct tat ggc ctt tac ttg gtt cgc cgc aac ggc 931

Phe Gly Ala Ala Ile Ala Tyr Gly Leu Tyr Leu Val Arg Arg Asn Gly

265 270 275

agc att gat cca gat gca acc gct gct cca gtg cct gca gga acg acc 979

Ser Ile Asp Pro Asp Ala Thr Ala Ala Pro Val Pro Ala Gly Thr Thr

280 285 290

aaa gcc gaa gca gaa gca ccc gca gaa ttt tca aac gat tcc acc atc 1027

Lys Ala Glu Ala Glu Ala Pro Ala Glu Phe Ser Asn Asp Ser Thr Ile

295 300 305

atc cag gca cct ttg acc ggt gaa gct att gca ctg agc agc gtc agc 1075

Ile Gln Ala Pro Leu Thr Gly Glu Ala Ile Ala Leu Ser Ser Val Ser

310 315 320 325

gat gcc atg ttt gcc agc gga aag ctt ggc tcg ggc gtt gcc atc gtc 1123

Asp Ala Met Phe Ala Ser Gly Lys Leu Gly Ser Gly Val Ala Ile Val

330 335 340

cca acc aag ggg cag tta gtt tct ccg gtg agt gga aag att gtg gtg 1171

Pro Thr Lys Gly Gln Leu Val Ser Pro Val Ser Gly Lys Ile Val Val

345 350 355

gca ttc cca tct ggc cat gct ttc gca gtt cgc acc aag gct gag gat 1219

Ala Phe Pro Ser Gly His Ala Phe Ala Val Arg Thr Lys Ala Glu Asp

360 365 370

ggt tcc aat gtg gat atc ttg atg cac att ggt ttc gac aca gta aac 1267

Gly Ser Asn Val Asp Ile Leu Met His Ile Gly Phe Asp Thr Val Asn

375 380 385

ctc aac ggc acg cac ttt aac ccg ctg aag aag cag ggc gat gaa gtc 1315

Leu Asn Gly Thr His Phe Asn Pro Leu Lys Lys Gln Gly Asp Glu Val

390 395 400 405

aaa gca ggg gag ctg ctg tgt gaa ttc gat att gat gcc att aag gct 1363

Lys Ala Gly Glu Leu Leu Cys Glu Phe Asp Ile Asp Ala Ile Lys Ala

410 415 420

gca ggt tat gag gta acc acg ccg att gtt gtt tcg aat tac aag aaa 1411

Ala Gly Tyr Glu Val Thr Thr Pro Ile Val Val Ser Asn Tyr Lys Lys

425 430 435

acc gga cct gta aac act tac ggt ttg ggc gaa att gaa gcg gga gcc 1459

Thr Gly Pro Val Asn Thr Tyr Gly Leu Gly Glu Ile Glu Ala Gly Ala

440 445 450

aac ctg ctc aac gtc gca aag aaa gaa gcg gtg cca gca aca cca 1504

Asn Leu Leu Asn Val Ala Lys Lys Glu Ala Val Pro Ala Thr Pro

455 460 465

taagttgaaa ccttgagtgt tcg 1527

<210>2

<211>468

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>2

Met Ala Met Val Phe Pro Ser Leu Val Asn Gly Tyr Asp Val Ala Ala

1 5 10 15

Thr Met Ala Ala Gly Glu Met Pro Met Trp Ser Leu Phe Gly Leu Asp

20 25 30

Val Ala Gln Ala Gly Tyr Gln Gly Thr Val Leu Pro Val Leu Val Val

35 40 45

Ser Trp Ile Leu Ala Thr Ile Glu Lys Phe Leu His Lys Arg Leu Lys

50 55 60

Gly Thr Ala Asp Phe Leu Ile Thr Pro Val Leu Thr Leu Leu Leu Thr

65 70 75 80

Gly Phe Leu Thr Phe Ile Ala Ile Gly Pro Ala Met Arg Trp Val Gly

85 90 95

Asp Val Leu Ala His Gly Leu Gln Gly Leu Tyr Asp Phe Gly Gly Pro

100 105 110

Val Gly Gly Leu Leu Phe Gly Leu Val Tyr Ser Pro Ile Val Ile Thr

115 120 125

Gly Leu His Gln Ser Phe Pro Pro Ile Glu Leu Glu Leu Phe Asn Gln

130 135 140

Gly Gly Ser Phe Ile Phe Ala Thr Ala Ser Met Ala Asn Ile Ala Gln

145 150 155 160

Gly Ala Ala Cys Leu Ala Val Phe Phe Leu Ala Lys Ser Glu Lys Leu

165 170 175

Lys Gly Leu Ala Gly Ala Ser Gly Val Ser Ala Val Leu Gly Ile Thr

180 185 190

Glu Pro Ala Ile Phe Gly Val Asn Leu Arg Leu Arg Trp Pro Phe Phe

195 200 205

Ile Gly Ile Gly Thr Ala Ala Ile Gly Gly Ala Leu Ile Ala Leu Phe

210 215 220

Asn Ile Lys Ala Val Ala Leu Gly Ala Ala Gly Phe Leu Gly Val Val

225 230 235 240

Ser Ile Asp Ala Pro Asp Met Val Met Phe Leu Val Cys Ala Val Val

245 250 255

Thr Phe Phe Ile Ala Phe Gly Ala Ala Ile Ala Tyr Gly Leu Tyr Leu

260 265 270

Val Arg Arg Asn Gly Ser Ile Asp Pro Asp Ala Thr Ala Ala Pro Val

275 280 285

Pro Ala Gly Thr Thr Lys Ala Glu Ala Glu Ala Pro Ala Glu Phe Ser

290 295 300

Asn Asp Ser Thr Ile Ile Gln Ala Pro Leu Thr Gly Glu Ala Ile Ala

305 310 315 320

Leu Ser Ser Val Ser Asp Ala Met Phe Ala Ser Gly Lys Leu Gly Ser

325 330 335

Gly Val Ala Ile Val Pro Thr Lys Gly Gln Leu Val Ser Pro Val Ser

340 345 350

Gly Lys Ile Val Val Ala Phe Pro Ser Gly His Ala Phe Ala Val Arg

355 360 365

Thr Lys Ala Glu Asp Gly Ser Asn Val Asp Ile Leu Met His Ile Gly

370 375 380

Phe Asp Thr Val Asn Leu Asn Gly Thr His Phe Asn Pro Leu Lys Lys

385 390 395 400

Gln Gly Asp Glu Val Lys Ala Gly Glu Leu Leu Cys Glu Phe Asp Ile

405 410 415

Asp Ala Ile Lys Ala Ala Gly Tyr Glu Val Thr Thr Pro Ile Val Val

420 425 430

Ser Asn Tyr Lys Lys Thr Gly Pro Val Asn Thr Tyr Gly Leu Gly Glu

435 440 445

Ile Glu Ala Gly Ala Asn Leu Leu Asn Val Ala Lys Lys Glu Ala Val

450 455 460

Pro Ala Thr Pro

465

<210>3

<211>1109

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(1)..(1086)

<223>FRXA00315

<400>3

tat gat ttc ggc ggt cca gtc ggc ggt ctg ctc ttc ggt ctg gtc tac 48

Tyr Asp Phe Gly Gly Pro Val Gly Gly Leu Leu Phe Gly Leu Val Tyr

1 5 10 15

tca cca atc gtc atc act ggt ctg cac cag tcc ttc ccg cca att gag 96

Ser Pro Ile Val Ile Thr Gly Leu His Gln Ser Phe Pro Pro Ile Glu

20 25 30

ctg gag ctg ttt aac cag ggt gga tcc ttc atc rtc gca acg gca tct 144

Leu Glu Leu Phe Asn Gln Gly Gly Ser Phe Ile Phe Ala Thr Ala Ser

35 40 45

atg gct aat atc gcc cag ggt gcg gca tgt ttg gca gtg ttc ttc ctg 192

Met Ala Asn Ile Ala Gln Gly Ala Ala Cys Leu Ala Val Phe Phe Leu

50 55 60

gcg aag agt gaa aag ctc aag ggc ctt gca ggt gct tca ggt gtc tcc 240

Ala Lys Ser Glu Lys Leu Lys Gly Leu Ala Gly Ala Ser Gly Val Ser

65 70 75 80

gct gtt ctt ggt att acg gag cct gcg atc ttc ggt gtg aac ctt cgc 288

Ala Val Leu Gly Ile Thr Glu Pro Ala Ile Phe Gly Val Asn Leu Arg

85 90 95

ctg cgc tgg ccg ttc ttc atc ggt atc ggt acc gca gct atc ggt ggc 336

Leu Arg Trp Pro Phe Phe Ile Gly Ile Gly Thr Ala Ala Ile Gly Gly

100 105 110

gct ttg att gca ctc ttt aat atc aag gca gtt gcg ttg ggc gct gca 384

Ala Leu Ile Ala Leu Phe Asn Ile Lys Ala Val Ala Leu Gly Ala Ala

115 120 125

ggt ttc ttg ggt gtt gtt tct att gat gct cca gat atg gtc atg ttc 432

Gly Phe Leu Gly Val Val Ser Ile Asp Ala Pro Asp Met Val Met Phe

130 135 140

ttg gtg tgt gca gtt gtt acc ttc ttc atc gca ttc ggc gca gcg att 480

Leu Val Cys Ala Val Val Thr Phe Phe Ile Ala Phe Gly Ala Ala Ile

145 150 155 160

gct tat ggc ctt tac ttg gtt cgc cgc aac ggc agc att gat cca gat 528

Ala Tyr Gly Leu Tyr Leu Val Arg Arg Asn Gly Ser Ile Asp Pro Asp

165 170 175

gca acc gct gct cca gtg cct gca gga acg acc aaa gcc gaa gca gaa 576

Ala Thr Ala Ala Pro Val Pro Ala Gly Thr Thr Lys Ala Glu Ala Glu

180 185 190

gca ccc gca gaa ttt tca aac gat tcc acc atc atc cag gca cct ttg 624

Ala Pro Ala Glu Phe Ser Asn Asp Ser Thr Ile Ile Gln Ala Pro Leu

195 200 205

acc ggt gaa gct att gca ctg agc agc gtc agc gat gcc atg ttt gcc 672

Thr Gly Glu Ala Ile Ala Leu Ser Ser Val Ser Asp Ala Met Phe Ala

210 215 220

agc gga aag ctt ggc tcg ggc gtt gcc atc gtc cca acc aag ggg cag 720

Ser Gly Lys Leu Gly Ser Gly Val Ala Ile Val Pro Thr Lys Gly Gln

225 230 235 240

tta gtt tct ccg gtg agt gga aag att gtg gtg gca ttc cca tct ggc 768

Leu Val Ser Pro Val Ser Gly Lys Ile Val Val Ala Phe Pro Ser Gly

245 250 255

cat gct ttc gca gtt cgc acc aag gct gag gat ggt tcc aat gtg gat 816

His Ala Phe Ala Val Arg Thr Lys Ala Glu Asp Gly Ser Asn Val Asp

260 265 270

atc ttg atg cac att ggt ttc gac aca gta aac ctc aac ggc acg cac 864

Ile Leu Met His Ile Gly Phe Asp Thr Val Asn Leu Asn Gly Thr His

275 280 285

ttt aac ccg ctg aag aag cag ggc gat gaa gtc aaa gca ggg gag ctg 912

Phe Asn Pro Leu Lys Lys Gln Gly Asp Glu Val Lys Ala Gly Glu Leu

290 295 300

ctg tgt gaa ttc gat att gat gcc att aag gct gca ggt tat gag gta 960

Leu Cys Glu Phe Asp Ile Asp Ala Ile Lys Ala Ala Gly Tyr Glu Val

305 310 315 320

acc acg ccg att gtt gtt tcg aat tac aag aaa acc gga cct gta aac 1008

Thr Thr Pro Ile Val Val Ser Asn Tyr Lys Lys Thr Gly Pro Val Asn

325 330 335

act tac ggt ttg ggc gaa att gaa gcg gga gcc aac ctg ctc aac gtc 1056

Thr Tyr Gly Leu Gly Glu Ile Glu Ala Gly Ala Asn Leu Leu Asn Val

340 345 350

gca aag aaa gaa gcg gtg cca gca aca cca taagttgaaa ccttgagtgt 1106

Ala Lys Lys Glu Ala Val Pro Ala Thr Pro

355 360

tcg 1109

<210>4

<211>362

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>4

Tyr Asp Phe Gly Gly Pro Val Gly Gly Leu Leu Phe Gly Leu Val Tyr

1 5 10 15

Ser Pro Ile Val Ile Thr Gly Leu His Gln Ser Phe Pro Pro Ile Glu

20 25 30

Leu Glu Leu Phe Asn Gln Gly Gly Ser Phe Ile Phe Ala Thr Ala Ser

35 40 45

Met Ala Asn Ile Ala Gln Gly Ala Ala Cys Leu Ala Val Phe Phe Leu

50 55 60

Ala Lys Ser Glu Lys Leu Lys Gly Leu Ala Gly Ala Ser Gly Val Ser

65 70 75 80

Ala Val Leu Gly Ile Thr Glu Pro Ala Ile Phe Gly Val Asn Leu Arg

85 90 95

Leu Arg Trp Pro Phe Phe Ile Gly Ile Gly Thr Ala Ala Ile Gly Gly

100 105 110

Ala Leu Ile Ala Leu Phe Asn Ile Lys Ala Val Ala Leu Gly Ala Ala

115 120 125

Gly Phe Leu Gly Val Val Ser Ile Asp Ala Pro Asp Met Val Met Phe

130 135 140

Leu Val Cys Ala Val Val Thr Phe Phe Ile Ala Phe Gly Ala Ala Ile

145 150 155 160

Ala Tyr Gly Leu Tyr Leu Val Arg Arg Asn Gly Ser Ile Asp Pro Asp

165 170 175

Ala Thr Ala Ala Pro Val Pro Ala Gly Thr Thr Lys Ala Glu Ala Glu

180 185 190

Ala Pro Ala Glu Phe Ser Asn Asp Ser Thr Ile Ile Gln Ala Pro Leu

195 200 205

Thr Gly Glu Ala Ile Ala Leu Ser Ser Val Ser Asp Ala Met Phe Ala

210 215 220

Ser Gly Lys Leu Gly Ser Gly Val Ala Ile Val Pro Thr Lys Gly Gln

225 230 235 240

Leu Val Ser Pro Val Ser Gly Lys Ile Val Val Ala Phe Pro Ser Gly

245 250 255

His Ala Phe Ala Val Arg Thr Lys Ala Glu Asp Gly Ser Asn Val Asp

260 265 270

Ile Leu Met His Ile Gly Phe Asp Thr Val Asn Leu Asn Gly Thr His

275 280 285

Phe Asn Pro Leu Lys Lys Gln Gly Asp Glu Val Lys Ala Gly Glu Leu

290 295 300

Leu Cys Glu Phe Asp Ile Asp Ala Ile Lys Ala Ala Gly Tyr Glu Val

305 310 315 320

Thr Thr Pro Ile Val Val Ser Asn Tyr Lys Lys Thr Gly Pro Val Asn

325 330 335

Thr Tyr Gly Leu Gly Glu Ile Glu Ala Gly Ala Asn Leu Leu Asn Val

340 345 350

Ala Lys Lys Glu Ala Val Pro Ala Thr Pro

355 360

<210>5

<211>372

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(349)

<223>RXA01503

<400>5

gtatcctcaa aggccttcta gctgttgcag ctgcagcgca ctcggtggat acgacatcca 60

cgacctatca aattctttat gctgcaggcg atgccttttc atg ttc ttg gca gtc 115

Met Phe Leu Ala Val

1 5

att ttg gcg att act gcg gct cgt aaa ttc ggt gcc aat gtc ttt aca 163

Ile Leu Ala Ile Thr Ala Ala Arg Lys Phe Gly Ala Asn Val Phe Thr

10 15 20

tca gtc gca ctc gct ggt gca ttg ctg cac aca cag ctt cag gca gta 211

Ser Val Ala Leu Ala Gly Ala Leu Leu His Thr Gln Leu Gln Ala Val

25 30 35

acc gtg ttg gtt gac ggt gaa ctc cag tcg atg act ctg gtg gct ttc 259

Thr Val Leu Val Asp Gly Glu Leu Gln Ser Met Thr Leu Val Ala Phe

40 45 50

caa aag gct ggt aat gac gtc acc ttc ctg ggc att cca gtg gtg ctg 307

Gln Lys Ala Gly Asn Asp Val Thr Phe Leu Gly Ile Pro Val Val Leu

55 60 65

cag ttg gcg ttg cat gta gcg agt ttg atg aag ttg tcg cga 349

Gln Leu Ala Leu His Val Ala Ser Leu Met Lys Leu Ser Arg

70 75 80

taagaggagg ggcgtgtcgg tct 372

<210>6

<211>83

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>6

Met Phe Leu Ala Val Ile Leu Ala Ile Thr Ala Ala Arg Lys Phe Gly

1 5 10 15

Ala Asn Val Phe Thr Ser Val Ala Leu Ala Gly Ala Leu Leu His Thr

20 25 30

Gln Leu Gln Ala Val Thr Val Leu Val Asp Gly Glu Leu Gln Ser Met

35 40 45

Thr Leu Val Ala Phe Gln Lys Ala Gly Asn Asp Val Thr Phe Leu Gly

50 55 60

Ile Pro Val Val Leu Gln Leu Ala Leu His Val Ala Ser Leu Met Lys

65 70 75 80

Leu Ser Arg

<210>7

<211>2187

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(2164)

<223>RXN01299

<400>7

cgactgcggc gtctcttcct ggcactacca ttcctcgtcc tgaccaactc gccacagctg 60

gtgcaacggt cacccaagtc aaaggattga aagaatcagc atg aat agc gta aat 115

Met Asn Ser Val Asn

1 5

aat tcc tcg ctt gtc cgg ctg gat gtc gat ttc ggc gac tcc acc acg 163

Asn Ser Ser Leu Val Arg Leu Asp Val Asp Phe Gly Asp Ser Thr Thr

10 15 20

gat gtc atc aac aac ctt gcc act gtt att ttc gac gct ggc cga gct 211

Asp Val Ile Asn Asn Leu Ala Thr Val Ile Phe Asp Ala Gly Arg Ala

25 30 35

tcc tcc gcc gac gcc ctt gcc aaa gac gcg ctg gat cgt gaa gca aag 259

Ser Ser Ala Asp Ala Leu Ala Lys Asp Ala Leu Asp Arg Glu Ala Lys

40 45 50

tcc ggc acc ggc gtt cct ggt caa gtt gct atc ccc cac tgc cgt tcc 307

Ser Gly Thr Gly Val Pro Gly Gln Val Ala Ile Pro His Cys Arg Ser

55 60 65

gaa gcc gta tct gtc cct acc ttg ggc ttt gct cgc ctg agc aag ggt 355

Glu Ala Val Ser Val Pro Thr Leu Gly Phe Ala Arg Leu Ser Lys Gly

70 75 80 85

gtg gac ttc agc gga cct gat ggc gat gcc aac ttg gtg ttc ctc att 403

Val Asp Phe Ser Gly Pro Asp Gly Asp Ala Asn Leu Val Phe Leu Ile

90 95 100

gca gca cct gct ggc ggc ggc aaa gag cac ctg aag atc ctg tcc aag 451

Ala Ala Pro Ala Gly Gly Gly Lys Glu His Leu Lys Ile Leu Ser Lys

105 110 115

ctt gct cgc tcc ttg gtg aag aag gat ttc atc aag gct ctg cag gaa 499

Leu Ala Arg Ser Leu Val Lys Lys Asp Phe Ile Lys Ala Leu Gln Glu

120 125 130

gcc acc acc gag cag gaa atc gtc gac gtt gtc gat gcc gtg ctc aac 547

Ala Thr Thr Glu Gln Glu Ile Val Asp Val Val Asp Ala Val Leu Asn

135 140 145

cca gca cca aaa acc acc gag cca gct gca gct ccg gct gcg gcg gcg 595

Pro Ala Pro Lys Thr Thr Glu Pro Ala Ala Ala Pro Ala Ala Ala Ala

150 155 160 165

gtt gct gag agt ggg gcg gcg tcg aca agc gtt act cgt atc gtg gca 643

Val Ala Glu Ser Gly Ala Ala Ser Thr Ser Val Thr Arg Ile Val Ala

170 175 180

atc acc gca tgc cca acc ggt atc gca cac acc tac atg gct gcg gat 691

Ile Thr Ala Cys Pro Thr Gly Ile Ala His Thr Tyr Met Ala Ala Asp

185 190 195

tcc ctg acg caa aac gcg gaa ggc cgc gat gat gtg gaa ctc gtt gtg 739

Ser Leu Thr Gln Asn Ala Glu Gly Arg Asp Asp Val Glu Leu Val Val

200 205 210

gag act cag ggc tct tcc gct gtc acc cca gtc gat ccg aag atc atc 787

Glu Thr Gln Gly Ser Ser Ala Val Thr Pro Val Asp Pro Lys Ile Ile

215 220 225

gaa gct gcc gac gcc gtc atc ttc gcc acc gac gtg gga gtt aaa gac 835

Glu Ala Ala Asp Ala Val Ile Phe Ala Thr Asp Val Gly Val Lys Asp

230 235 240 245

cgc gag cgt ttc gct ggc aag cca gtc att gaa tcc ggc gtc aag cgc 883

Arg Glu Arg Phe Ala Gly Lys Pro Val Ile Glu Ser Gly Val Lys Arg

250 255 260

gcg atc aat gag cca gcc aag atg atc gac gag gcc atc gca gcc tcc 931

Ala Ile Asn Glu Pro Ala Lys Met Ile Asp Glu Ala Ile Ala Ala Ser

265 270 275

aag aac cca aac gcc cgc aag gtt tcc ggt tcc ggt gtc gcg gca tct 979

Lys Asn Pro Asn Ala Arg Lys Val Ser Gly Ser Gly Val Ala Ala Ser

280 285 290

gct gaa acc acc ggc gag aag ctc ggc tgg ggc aag cgc atc cag cag 1027

Ala Glu Thr Thr Gly Glu Lys Leu Gly Trp Gly Lys Arg Ile Gln Gln

295 300 305

gca gtc atg acc ggc gtg tcc tac atg gtt cca ttc gta gct gcc ggc 1075

Ala Val Met Thr Gly Val Ser Tyr Met Val Pro Phe Val Ala Ala Gly

310 315 320 325

ggc ctc ctg ttg gct ctc ggc ttc gca ttc ggt gga tac gac atg gcg 1123

Gly Leu Leu Leu Ala Leu Gly Phe Ala Phe Gly Gly Tyr Asp Met Ala

330 335 340

aac ggc tgg caa gca atc gcc acc cag ttc tct ctg acc aac ctg cca 1171

Asn Gly Trp Gln Ala Ile Ala Thr Gln Phe Ser Leu Thr Asn Leu Pro

345 350 355

ggc aac acc gtc gat gtt gac ggc gtg gcc atg acc ttc gag cgt tca 1219

Gly Asn Thr Val Asp Val Asp Gly Val Ala Met Thr Phe Glu Arg Ser

360 365 370

ggc ttc ctg ttg tac ttc ggc gca gtc ctg ttc gcc acc ggc caa gca 1267

Gly Phe Leu Leu Tyr Phe Gly Ala Val Leu Phe Ala Thr Gly Gln Ala

375 380 385

gcc atg ggc ttc atc gtg gca gcc ctg tct ggc tac acc gca tac gca 1315

Ala Met Gly Phe Ile Val Ala Ala Leu Ser Gly Tyr Thr Ala Tyr Ala

390 395 400 405

ctt gct gga cgc cca ggc atc gcg ccg ggc ttc gtc ggt ggc gcc atc 1363

Leu Ala Gly Arg Pro Gly Ile Ala Pro Gly Phe Val Gly Gly Ala Ile

410 415 420

tcc gtc acc atc ggc gct ggc ttc att ggt ggt ctg gtt acc ggt atc 1411

Ser Val Thr Ile Gly Ala Gly Phe Ile Gly Gly Leu Val Thr Gly Ile

425 430 435

ttg gct ggt ctc att gcc ctg tgg att ggc tcc tgg aag gtg cca cgc 1459

Leu Ala Gly Leu Ile Ala Leu Trp Ile Gly Ser Trp Lys Val Pro Arg

440 445 450

gtg gtg cag tca ctg atg cct gtg gtc atc atc ccg cta ctt acc tca 1507

Val Val Gln Ser Leu Met Pro Val Val Ile Ile Pro Leu Leu Thr Ser

455 460 465

gtg gtt gtt ggt ctc gtc atg tac ctc ctg ctg ggt cgc cca ctc gca 1555

Val Val Val Gly Leu Val Met Tyr Leu Leu Leu Gly Arg Pro Leu Ala

470 475 480 485

tcc atc atg act ggt ttg cag gac tgg cta tcg tca atg tcc gga agc 1603

Ser Ile Met Thr Gly Leu Gln Asp Trp Leu Ser Ser Met Ser Gly Ser

490 495 500

tcc gcc atc ttg ctg ggt atc atc ttg ggc ctc atg atg tgt ttc gac 1651

Ser Ala Ile Leu Leu Gly Ile Ile Leu Gly Leu Met Met Cys Phe Asp

505 510 515

ctc ggc gga cca gta aac aag gca gcc tac ctc ttt ggt acc gca ggc 1699

Leu Gly Gly Pro Val Asn Lys Ala Ala Tyr Leu Phe Gly Thr Ala Gly

520 525 530

ctg tct acc ggc gac caa gct tcc atg gaa atc atg gcc gcg atc atg 1747

Leu Ser Thr Gly Asp Gln Ala Ser Met Glu Ile Met Ala Ala Ile Met

535 540 545

gca gct ggc atg gtc cca cca atc gcg ttg tcc att gct acc ctg ctg 1795

Ala Ala Gly Met Val Pro Pro Ile Ala Leu Ser Ile Ala Thr Leu Leu

550 555 560 565

cgc aag aag ctg ttc acc cca gca gag caa gaa aac ggc aag tct tcc 1843

Arg Lys Lys Leu Phe Thr Pro Ala Glu Gln Glu Asn Gly Lys Ser Ser

570 575 580

tgg ctg ctt ggc ctg gca ttc gtc tcc gaa ggt gcc atc cca ttc gcc 1891

Trp Leu Leu Gly Leu Ala Phe Val Ser Glu Gly Ala Ile Pro Phe Ala

585 590 595

gca gct gac cca ttc cgt gtg atc cca gca atg atg gct ggc ggt gca 1939

Ala Ala Asp Pro Phe Arg Val Ile Pro Ala Met Met Ala Gly Gly Ala

600 605 610

acc act ggt gca atc tcc atg gca ctg ggc gtc ggc tct cgg gct cca 1987

Thr Thr Gly Ala Ile Ser Met Ala Leu Gly Val Gly Ser Arg Ala Pro

615 620 625

cac ggc ggt atc ttc gtg gtc tgg gca atc gaa cca tgg tgg ggc tgg 2035

His Gly Gly Ile Phe Val Val Trp Ala Ile Glu Pro Trp Trp Gly Trp

630 635 640 645

ctc atc gca ctt gca gca ggc acc atc gtg tcc acc atc gtt gtc atc 2083

Leu Ile Ala Leu Ala Ala Gly Thr Ile Val Ser Thr Ile Val Val Ile

650 655 660

gca ctg aag cag ttc tgg cca aac aag gcc gtc gct gca gaa gtc gcg 2131

Ala Leu Lys Gln Phe Trp Pro Asn Lys Ala Val Ala Ala Glu Val Ala

665 670 675

aag caa gaa gca caa caa gca gct gta aac gca taatcggacc ttgacccgat 2184

Lys Gln Glu Ala Gln Gln Ala Ala Val Asn Ala

680 685

gtc 2187

<210>8

<211>688

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>8

Met Asn Ser Val Asn Asn Ser Ser Leu Val Arg Leu Asp Val Asp Phe

1 5 10 15

Gly Asp Ser Thr Thr Asp Val Ile Asn Asn Leu Ala Thr Val Ile Phe

20 25 30

Asp Ala Gly Arg Ala Ser Ser Ala Asp Ala Leu Ala Lys Asp Ala Leu

35 40 45

Asp Arg Glu Ala Lys Ser Gly Thr Gly Val Pro Gly Gln Val Ala Ile

50 55 60

Pro His Cys Arg Ser Glu Ala Val Ser Val Pro Thr Leu Gly Phe Ala

65 70 75 80

Arg Leu Ser Lys Gly Val Asp Phe Ser Gly Pro Asp Gly Asp Ala Asn

85 90 95

Leu Val Phe Leu Ile Ala Ala Pro Ala Gly Gly Gly Lys Glu His Leu

100 105 110

Lys Ile Leu Ser Lys Leu Ala Arg Ser Leu Val Lys Lys Asp Phe Ile

115 120 125

Lys Ala Leu Gln Glu Ala Thr Thr Glu Gln Glu Ile Val Asp Val Val

130 135 140

Asp Ala Val Leu Asn Pro Ala Pro Lys Thr Thr Glu Pro Ala Ala Ala

145 150 155 160

Pro Ala Ala Ala Ala Val Ala Glu Ser Gly Ala Ala Ser Thr Ser Val

165 170 175

Thr Arg Ile Val Ala Ile Thr Ala Cys Pro Thr Gly Ile Ala His Thr

180 185 190

Tyr Met Ala Ala Asp Ser Leu Thr Gln Asn Ala Glu Gly Arg Asp Asp

195 200 205

Val Glu Leu Val Val Glu Thr Gln Gly Ser Ser Ala Val Thr Pro Val

210 215 220

Asp Pro Lys Ile Ile Glu Ala Ala Asp Ala Val Ile Phe Ala Thr Asp

225 230 235 240

Val Gly Val Lys Asp Arg Glu Arg Phe Ala Gly Lys Pro Val Ile Glu

245 250 255

Ser Gly Val Lys Arg Ala Ile Asn Glu Pro Ala Lys Met Ile Asp Glu

260 265 270

Ala Ile Ala Ala Ser Lys Asn Pro Asn Ala Arg Lys Val Ser Gly Ser

275 280 285

Gly Val Ala Ala Ser Ala Glu Thr Thr Gly Glu Lys Leu Gly Trp Gly

290 295 300

Lys Arg Ile Gln Gln Ala Val Met Thr Gly Val Ser Tyr Met Val Pro

305 310 315 320

Phe Val Ala Ala Gly Gly Leu Leu Leu Ala Leu Gly Phe Ala Phe Gly

325 330 335

Gly Tyr Asp Met Ala Asn Gly Trp Gln Ala Ile Ala Thr Gln Phe Ser

340 345 350

Leu Thr Asn Leu Pro Gly Asn Thr Val Asp Val Asp Gly Val Ala Met

355 360 365

Thr Phe Glu Arg Ser Gly Phe Leu Leu Tyr Phe Gly Ala Val Leu Phe

370 375 380

Ala Thr Gly Gln Ala Ala Met Gly Phe Ile Val Ala Ala Leu Ser Gly

385 390 395 400

Tyr Thr Ala Tyr Ala Leu Ala Gly Arg Pro Gly Ile Ala Pro Gly Phe

405 410 415

Val Gly Gly Ala Ile Ser Val Thr Ile Gly Ala Gly Phe Ile Gly Gly

420 425 430

Leu Val Thr Gly Ile Leu Ala Gly Leu Ile Ala Leu Trp Ile Gly Ser

435 440 445

Trp Lys Val Pro Arg Val Val Gln Ser Leu Met Pro Val Val Ile Ile

450 455 460

Pro Leu Leu Thr Ser Val Val Val Gly Leu Val Met Tyr Leu Leu Leu

465 470 475 480

Gly Arg Pro Leu Ala Ser Ile Met Thr Gly Leu Gln Asp Trp Leu Ser

485 490 495

Ser Met Ser Gly Ser Ser Ala Ile Leu Leu Gly Ile Ile Leu Gly Leu

500 505 510

Met Met Cys Phe Asp Leu Gly Gly Pro Val Asn Lys Ala Ala Tyr Leu

515 520 525

Phe Gly Thr Ala Gly Leu Ser Thr Gly Asp Gln Ala Ser Met Glu Ile

530 535 540

Met Ala Ala Ile Met Ala Ala Gly Met Val Pro Pro Ile Ala Leu Ser

545 550 555 560

Ile Ala Thr Leu Leu Arg Lys Lys Leu Phe Thr Pro Ala Glu Gln Glu

565 570 575

Asn Gly Lys Ser Ser Trp Leu Leu Gly Leu Ala Phe Val Ser Glu Gly

580 585 590

Ala Ile Pro Phe Ala Ala Ala Asp Pro Phe Arg Val Ile Pro Ala Met

595 600 605

Met Ala Gly Gly Ala Thr Thr Gly Ala Ile Ser Met Ala Leu Gly Val

610 615 620

Gly Ser Arg Ala Pro His Gly Gly Ile Phe Val Val Trp Ala Ile Glu

625 630 635 640

Pro Trp Trp Gly Trp Leu Ile Ala Leu Ala Ala Gly Thr Ile Val Ser

645 650 655

Thr Ile Val Val Ile Ala Leu Lys Gln Phe Trp Pro Asn Lys Ala Val

660 665 670

Ala Ala Glu Val Ala Lys Gln Glu Ala Gln Gln Ala Ala Val Asn Ala

675 680 685

<210>9

<211>464

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(1)..(441)

<223>FRXA01299

<400>9

atg gaa atc atg gcc gcg atc atg gca gct ggc atg gtc cca cca atc 48

Met Glu Ile Met Ala Ala Ile Met Ala Ala Gly Met Val Pro Pro Ile

1 5 10 15

gcg ttg tcc att gct acc ctg ctg cgc aag aag ctg ttc acc cca gca 96

Ala Leu Ser Ile Ala Thr Leu Leu Arg Lys Lys Leu Phe Thr Pro Ala

20 25 30

gag caa gaa aac ggc aag tct tcc tgg ctg ctt ggc ctg gca ttc gtc 144

Glu Gln Glu Asn Gly Lys Ser Ser Trp Leu Leu Gly Leu Ala Phe Val

35 40 45

tcc gaa ggt gcc atc cca ttc gcc gca gct gac cca ttc cgt gtg atc 192

Ser Glu Gly Ala Ile Pro Phe Ala Ala Ala Asp Pro Phe Arg Val Ile

50 55 60

cca gca atg atg gct ggc ggt gca acc act ggt gca atc tcc atg gca 240

Pro Ala Met Met Ala Gly Gly Ala Thr Thr Gly Ala Ile Ser Met Ala

65 70 75 80

ctg ggc gtc ggc tct cgg gct cca cac ggc ggt atc ttc gtg gtc tgg 288

Leu Gly Val Gly Ser Arg Ala Pro His Gly Gly Ile Phe Val Val Trp

85 90 95

gca atc gaa cca tgg tgg ggc tgg ctc atc gca ctt gca gca ggc acc 336

Ala Ile Glu Pro Trp Trp Gly Trp Leu Ile Ala Leu Ala Ala Gly Thr

100 105 110

atc gtg tcc acc atc gtt gtc atc gca ctg aag cag ttc tgg cca aac 384

Ile Val Ser Thr Ile Val Val Ile Ala Leu Lys Gln Phe Trp Pro Asn

115 120 125

aag gcc gtc gct gca gaa gtc gcg aag caa gaa gca caa caa gca gct 432

Lys Ala Val Ala Ala Glu Val Ala Lys Gln Glu Ala Gln Gln Ala Ala

130 135 140

gta aac gca taatcggacc ttgacccgat gtc 464

Val Asn Ala

145

<210>10

<211>147

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>10

Met Glu Ile Met Ala Ala Ile Met Ala Ala Gly Met Val Pro Pro Ile

1 5 10 15

Ala Leu Ser Ile Ala Thr Leu Leu Arg Lys Lys Leu Phe Thr Pro Ala

20 25 30

Glu Gln Glu Asn Gly Lys Ser Ser Trp Leu Leu Gly Leu Ala Phe Val

35 40 45

Ser Glu Gly Ala Ile Pro Phe Ala Ala Ala Asp Pro Phe Arg Val Ile

50 55 60

Pro Ala Met Met Ala Gly Gly Ala Thr Thr Gly Ala Ile Ser Met Ala

65 70 75 80

Leu Gly Val Gly Ser Arg Ala Pro His Gly Gly Ile Phe Val Val Trp

85 90 95

Ala Ile Glu Pro Trp Trp Gly Trp Leu Ile Ala Leu Ala Ala Gly Thr

100 105 110

Ile Val Ser Thr Ile Val Val Ile Ala Leu Lys Gln Phe Trp Pro Asn

115 120 125

Lys Ala Val Ala Ala Glu Val Ala Lys Gln Glu Ala Gln Gln Ala Ala

130 135 140

Val Asn Ala

145

<210>11

<211>580

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(580)

<223>FRXA01883

<400>11

cgactgcggc gtctcttcct ggcactacca ttcctcgtcc tgaccaactc gccacagctg 60

gtgcaacggt cacccaagtc aaaggattga aagaatcagc atg aat agc gta aat 115

Met Asn Ser Val Asn

1 5

aat tcc tcg ctt gtc cgg ctg gat gtc gat ttc ggc gac tcc acc acg 163

Asn Ser Ser Leu Val Arg Leu Asp Val Asp Phe Gly Asp Ser Thr Thr

10 15 20

gat gtc atc aac aac ctt gcc act gtt att ttc gac gct ggc cga gct 211

Asp Val Ile Asn Asn Leu Ala Thr Val Ile Phe Asp Ala Gly Arg Ala

25 30 35

tcc tcc gcc gac gcc ctt gcc aaa gac gcg ctg gat cgt gaa gca aag 259

Ser Ser Ala Asp Ala Leu Ala Lys Asp Ala Leu Asp Arg Glu Ala Lys

40 45 50

tcc ggc acc ggc gtt cct ggt caa gtt gct atc ccc cac tgc cgt tcc 307

Ser Gly Thr Gly Val Pro Gly Gln Val Ala Ile Pro His Cys Arg Ser

55 60 65

gaa gcc gta tct gtc cct acc ttg ggc ttt gct cgc ctg agc aag ggt 355

Glu Ala Val Ser Val Pro Thr Leu Gly Phe Ala Arg Leu Ser Lys Gly

70 75 80 85

gtg gac ttc agc gga cct gat ggc gat gcc aac ttg gtg ttc ctc att 403

Val Asp Phe Ser Gly Pro Asp Gly Asp Ala Asn Leu Val Phe Leu Ile

90 95 100

gca gca cct gct ggc ggc ggc aaa gag cac ctg aag atc ctg tcc aag 451

Ala Ala Pro Ala Gly Gly Gly Lys Glu His Leu Lys Ile Leu Ser Lys

105 110 115

ctt gct cgc tcc ttg gtg aag aag gat ttc atc aag gct ctg cag gaa 499

Leu Ala Arg Ser Leu Val Lys Lys Asp Phe Ile Lys Ala Leu Gln Glu

120 125 130

gcc acc acc gag cag gaa atc gtc gac gtt gtc gat gcc gtg ctc aac 547

Ala Thr Thr Glu Gln Glu Ile Val Asp Val Val Asp Ala Val Leu Asn

135 140 145

cca gca cca aaa aac cac cga gcc agc tgc agc 580

Pro Ala Pro Lys Asn His Arg Ala Ser Cys Ser

150 155 160

<210>12

<211>160

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>12

Met Asn Ser Val Asn Asn Ser Ser Leu Val Arg Leu Asp Val Asp Phe

1 5 10 15

Gly Asp Ser Thr Thr Asp Val Ile Asn Asn Leu Ala Thr Val Ile Phe

20 25 30

Asp Ala Gly Arg Ala Ser Ser Ala Asp Ala Leu Ala Lys Asp Ala Leu

35 40 45

Asp Arg Glu Ala Lys Ser Gly Thr Gly Val Pro Gly Gln Val Ala Ile

50 55 60

Pro His Cys Arg Ser Glu Ala Val Ser Val Pro Thr Leu Gly Phe Ala

65 70 75 80

Arg Leu Ser Lys Gly Val Asp Phe Ser Gly Pro Asp Gly Asp Ala Asn

85 90 95

Leu Val Phe Leu Ile Ala Ala Pro Ala Gly Gly Gly Lys Glu His Leu

100 105 110

Lys Ile Leu Ser Lys Leu Ala Arg Ser Leu Val Lys Lys Asp Phe Ile

115 120 125

Lys Ala Leu Gln Glu Ala Thr Thr Glu Gln Glu Ile Val Asp Val Val

130 135 140

Asp Ala Val Leu Asn Pro Ala Pro Lys Asn His Arg Ala Ser Cys Ser

145 150 155 160

<210>13

<211>631

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(77)..(631)

<223>FRXA01889

<400>13

accgagccag ctgcagctcc ggctgcggcg gccggttgtt aagagtgggg cggcgtcgac 60

aagcgttact cgtatc gtg gca atc acc gca tgc cca acc ggt atc gca cac 112

Val Ala Ile Thr Ala Cys Pro Thr Gly Ile Ala His

1 5 10

acc tac atg gct gcg gat tcc ctg acg caa aac gcg gaa ggc cgc gat 160

Thr Tyr Met Ala Ala Asp Ser Leu Thr Gln Asn Ala Glu Gly Arg Asp

15 20 25

gat gtg gaa ctc gtt gtg gag act cag ggc tct tcc gct gtc acc cca 208

Asp Val Glu Leu Val Val Glu Thr Gln Gly Ser Ser Ala Val Thr Pro

30 35 40

gtc gat ccg aag atc atc gaa gct gcc gac gcc gtc atc ttc gcc acc 256

Val Asp Pro Lys Ile Ile Glu Ala Ala Asp Ala Val Ile Phe Ala Thr

45 50 55 60

gac gtg gga gtt aaa gac cgc gag cgt ttc gct ggc aag cca gtc att 304

Asp Val Gly Val Lys Asp Arg Glu Arg Phe Ala Gly Lys Pro Val Ile

65 70 75

gaa tcc ggc gtc aag cgc gcg atc aat gag cca gcc aag atg atc gac 352

Glu Ser Gly Val Lys Arg Ala Ile Asn Glu Pro Ala Lys Met Ile Asp

80 85 90

gag gcc atc gca gcc tcc aag aac cca aac gcc cgc aag gtt tcc ggt 400

Glu Ala Ile Ala Ala Ser Lys Asn Pro Asn Ala Arg Lys Val Ser Gly

95 100 105

tcc ggt gtc gcg gca tct gct gaa acc acc ggc gag aag ctc ggc tgg 448

Ser Gly Val Ala Ala Ser Ala Glu Thr Thr Gly Glu Lys Leu Gly Trp

110 115 120

ggc aag cgc atc cag cag gca gtc atg acc ggc gtg tcc tac atg gtt 496

Gly Lys Arg Ile Gln Gln Ala Val Met Thr Gly Val Ser Tyr Met Val

125 130 135 140

cca ttc gta gct gcc ggc ggc ctc ctg ttg gct ctc ggc ttc gca ttc 544

Pro Phe Val Ala Ala Gly Gly Leu Leu Leu Ala Leu Gly Phe Ala Phe

145 150 155

ggt gga tac gac atg gcg aac ggc tgg caa gca atc gcc acc cag ttc 592

Gly Gly Tyr Asp Met Ala Asn Gly Trp Gln Ala Ile Ala Thr Gln Phe

160 165 170

tct ctg acc aac ctg cca ggc aac acc gtc gat gtt gac 631

Ser Leu Thr Asn Leu Pro Gly Asn Thr Val Asp Val Asp

175 180 185

<210>14

<211>185

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>14

Val Ala Ile Thr Ala Cys Pro Thr Gly Ile Ala His Thr Tyr Met Ala

1 5 10 15

Ala Asp Ser Leu Thr Gln Asn Ala Glu Gly Arg Asp Asp Val Glu Leu

20 25 30

Val Val Glu Thr Gln Gly Ser Ser Ala Val Thr Pro Val Asp Pro Lys

35 40 45

Ile Ile Glu Ala Ala Asp Ala Val Ile Phe Ala Thr Asp Val Gly Val

50 55 60

Lys Asp Arg Glu Arg Phe Ala Gly Lys Pro Val Ile Glu Ser Gly Val

65 70 75 80

Lys Arg Ala Ile Asn Glu Pro Ala Lys Met Ile Asp Glu Ala Ile Ala

85 90 95

Ala Ser Lys Asn Pro Asn Ala Arg Lys Val Ser Gly Ser Gly Val Ala

100 105 110

Ala Ser Ala Glu Thr Thr Gly Glu Lys Leu Gly Trp Gly Lys Arg Ile

115 120 125

Gln Gln Ala Val Met Thr Gly Val Ser Tyr Met Val Pro Phe Val Ala

130 135 140

Ala Gly Gly Leu Leu Leu Ala Leu Gly Phe Ala Phe Gly Gly Tyr Asp

145 150 155 160

Met Ala Asn Gly Trp Gln Ala Ile Ala Thr Gln Phe Ser Leu Thr Asn

165 170 175

Leu Pro Gly Asn Thr Val Asp Val Asp

180 185

<210>15

<211>416

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(1)..(393)

<223>RXA00951

<400>15

atc caa gca atc tta gag aag gca gca gcg ccg gcg aag cag aag gct 48

Ile Gln Ala Ile Leu Glu Lys Ala Ala Ala Pro Ala Lys Gln Lys Ala

1 5 10 15

cct gct gtg gct cct gct gta aca ccc act gac gct cct gca gcc tca 96

Pro Ala Val Ala Pro Ala Val Thr Pro Thr Asp Ala Pro Ala Ala Ser

20 25 30

gtc caa tcc aaa acc cac gac aag atc ctc acc gtc tgt ggc aac ggc 144

Val Gln Ser Lys Thr His Asp Lys Ile Leu Thr Val Cys Gly Asn Gly

35 40 45

ttg ggt acc tcc ctc ttc ctc aaa aac acc ctt gag caa gtt ttc gac 192

Leu Gly Thr Ser Leu Phe Leu Lys Asn Thr Leu Glu Gln Val Phe Asp

50 55 60

acc tgg ggt tgg ggt cca tac atg acg gtg gag gca acc gac act atc 240

Thr Trp Gly Trp Gly Pro Tyr Met Thr Val Glu Ala Thr Asp Thr Ile

65 70 75 80

tcc gcc aag ggc aaa gcc aag gaa gct gat ctc atc atg acc tct ggt 288

Ser Ala Lys Gly Lys Ala Lys Glu Ala Asp Leu Ile Met Thr Ser Gly

85 90 95

gaa atc gcc cgc acg ttg ggt gat gtt gga atc ccg gtt cac gtg atc 336

Glu Ile Ala Arg Thr Leu Gly Asp Val Gly Ile Pro Val His Val Ile

100 105 110

aat gac ttc acg agc acc gat gaa atc gat gct gcg ctt cgt gaa cgc 384

Asn Asp Phe Thr Ser Thr Asp Glu Ile Asp Ala Ala Leu Arg Glu Arg

115 120 125

tac gac atc taactacttt aaaaggacga aaa 416

Tyr Asp Ile

130

<210>16

<211>131

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>16

Ile Gln Ala Ile Leu Glu Lys Ala Ala Ala Pro Ala Lys Gln Lys Ala

1 5 10 15

Pro Ala Val Ala Pro Ala Val Thr Pro Thr Asp Ala Pro Ala Ala Ser

20 25 30

Val Gln Ser Lys Thr His Asp Lys Ile Leu Thr Val Cys Gly Asn Gly

35 40 45

Leu Gly Thr Ser Leu Phe Leu Lys Asn Thr Leu Glu Gln Val Phe Asp

50 55 60

Thr Trp Gly Trp Gly Pro Tyr Met Thr Val Glu Ala Thr Asp Thr Ile

65 70 75 80

Ser Ala Lys Gly Lys Ala Lys Glu Ala Asp Leu Ile Met Thr Ser Gly

85 90 95

Glu Ile Ala Arg Thr Leu Gly Asp Val Gly Ile Pro Val His Val Ile

100 105 110

Asn Asp Phe Thr Ser Thr Asp Glu Ile Asp Ala Ala Leu Arg Glu Arg

115 120 125

Tyr Asp Ile

130

<210>17

<211>1827

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(1804)

<223>RXN01244

<400>17

gatatgtgtt tgtttgtcaa tatccaaatg tttgaatagt tgcacaactg ttggttttgt 60

ggtgatcttg aggaaattaa ctcaatgatt gtgaggatgg gtg gct act gtg gct 115

Val Ala Thr Val Ala

1 5

gat gtg aat caa gac act gta ctg aag ggc acc ggc gtt gtc ggt gga 163

Asp Val Asn Gln Asp Thr Val Leu Lys Gly Thr Gly Val Val Gly Gly

10 15 20

gtc cgt tat gca agc gcg gtg tgg att acc cca cgc ccc gaa cta ccc 211

Val Arg Tyr Ala Ser Ala Val Trp Ile Thr Pro Arg Pro Glu Leu Pro

25 30 35

caa gca ggc gaa gtc gtc gcc gaa gaa aac cgt gaa gca gag cag gag 259

Gln Ala Gly Glu Val Val Ala Glu Glu Asn Arg Glu Ala Glu Gln Glu

40 45 50

cgt ttc gac gcc gct gca gcc aca gtc tct tct cgt ttg ctt gag cgc 307

Arg Phe Asp Ala Ala Ala Ala Thr Val Ser Ser Arg Leu Leu Glu Arg

55 60 65

tcc gaa gct gct gaa gga cca gca gct gag gtg ctt aaa gct act gct 355

Ser Glu Ala Ala Glu Gly Pro Ala Ala Glu Val Leu Lys Ala Thr Ala

70 75 80 85

ggc atg gtc aat gac cgt ggc tgg cgt aag gct gtc atc aag ggt gtc 403

Gly Met Val Asn Asp Arg Gly Trp Arg Lys Ala Val Ile Lys Gly Val

90 95 100

aag ggt ggt cac cct gcg gaa tac gcc gtg gtt gca gca aca acc aag 451

Lys Gly Gly His Pro Ala Glu Tyr Ala Val Val Ala Ala Thr Thr Lys

105 110 115

ttc atc tcc atg ttc gaa gcc gca ggc ggc ctg atc gcg gag cgc acc 499

Phe Ile Ser Met Phe Glu Ala Ala Gly Gly Leu Ile Ala Glu Arg Thr

120 125 130

aca gac ttg cgc gac atc cgc gac cgc gtc atc gca gaa ctt cgt ggc 547

Thr Asp Leu Arg Asp Ile Arg Asp Arg Val Ile Ala Glu Leu Arg Gly

135 140 145

gat gaa gag cca ggt ctg cca gct gtt tcc gga cag gtc att ctc ttt 595

Asp Glu Glu Pro Gly Leu Pro Ala Val Ser Gly Gln Val Ile Leu Phe

150 155 160 165

gca gat gac ctc tcc cca gca gac acc gcg gca cta gac aca gat ctc 643

Ala Asp Asp Leu Ser Pro Ala Asp Thr Ala Ala Leu Asp Thr Asp Leu

170 175 180

ttt gtg gga ctt gtc act gag ctg ggt ggc cca acg agc cac acc gcg 691

Phe Val Gly Leu Val Thr Glu Leu Gly Gly Pro Thr Ser His Thr Ala

185 190 195

atc atc gca cgc cag ctc aac gtg cct tgc atc gtc gca tcc ggc gcc 739

Ile Ile Ala Arg Gln Leu Asn Val Pro Cys Ile Val Ala Ser Gly Ala

200 205 210

ggc atc aag gac atc aag tcc ggc gaa aag gtg ctt atc gac ggc agc 787

Gly Ile Lys Asp Ile Lys Ser Gly Glu Lys Val Leu Ile Asp Gly Ser

215 220 225

ctc ggc acc att gac cgc aac gcg gac gaa gct gaa gca acc aag ctc 835

Leu Gly Thr Ile Asp Arg Asn Ala Asp Glu Ala Glu Ala Thr Lys Leu

230 235 240 245

gtc tcc gag tcc ctc gag cgc gct gct cgc atc gcc gag tgg aag ggt 883

Val Ser Glu Ser Leu Glu Arg Ala Ala Arg Ile Ala Glu Trp Lys Gly

250 255 260

cct gca caa acc aag gac ggc tac cgc gtt cag ctg ttg gcc aac gtc 931

Pro Ala Gln Thr Lys Asp Gly Tyr Arg Val Gln Leu Leu Ala Asn Val

265 270 275

caa gac ggc aac tct gca cag cag gct gca cag acc gaa gca gaa ggc 979

Gln Asp Gly Asn Ser Ala Gln Gln Ala Ala Gln Thr Glu Ala Glu Gly

280 285 290

atc ggc ctg ttc cgc acc gaa ctg tgc ttc ctt tcc gcc acc gaa gag 1027

Ile Gly Leu Phe Arg Thr Glu Leu Cys Phe Leu Ser Ala Thr Glu Glu

295 300 305

cca agc gtt gat gag cag gct gcg gtc tac tca aag gtg ctt gaa gca 1075

Pro Ser Val Asp Glu Gln Ala Ala Val Tyr Ser Lys Val Leu Glu Ala

310 315 320 325

ttc cca gag tcc aag gtc gtt gtc cgc tcc ctc gac gca ggt tct gac 1123

Phe Pro Glu Ser Lys Val Val Val Arg Ser Leu Asp Ala Gly Ser Asp

330 335 340

aag cca gtt cca ttc gca tcg atg gct gat gag atg aac cca gca ctg 1171

Lys Pro Val Pro Phe Ala Ser Met Ala Asp Glu Met Asn Pro Ala Leu

345 350 355

ggt gtt cgt ggc ctg cgt atc gca cgt gga cag gtt gat ctg ctg act 1219

Gly Val Arg Gly Leu Arg Ile Ala Arg Gly Gln Val Asp Leu Leu Thr

360 365 370

cgc cag ctc gac gca att gcg aag gcc agc gaa gaa ctc ggc cgt ggc 1267

Arg Gln Leu Asp Ala Ile Ala Lys Ala Ser Glu Glu Leu Gly Arg Gly

375 380 385

gac gac gcc cca acc tgg gtt atg gct cca atg gtg gct acc gct tat 1315

Asp Asp Ala Pro Thr Trp Val Met Ala Pro Met Val Ala Thr Ala Tyr

390 395 400 405

gaa gca aag tgg ttt gct gac atg tgc cgt gag cgt ggc cta atc gcc 1363

Glu Ala Lys Trp Phe Ala Asp Met Cys Arg Glu Arg Gly Leu Ile Ala

410 415 420

ggc gcc atg atc gaa gtt cca gca gca tcc ctg atg gca gac aag atc 1411

Gly Ala Met Ile Glu Val Pro Ala Ala Ser Leu Met Ala Asp Lys Ile

425 430 435

atg cct cac ctg gac ttt gtt tcc atc ggt acc aac gac ctg acc cag 1459

Met Pro His Leu Asp Phe Val Ser Ile Gly Thr Asn Asp Leu Thr Gln

440 445 450

tac acc atg gca gcg gac cgc atg tct cct gag ctt gcc tac ctg acc 1507

Tyr Thr Met Ala Ala Asp Arg Met Ser Pro Glu Leu Ala Tyr Leu Thr

455 460 465

gat cct tgg cag cca gca gtc ctg cgc ctg atc aag cac acc tgt gac 1555

Asp Pro Trp Gln Pro Ala Val Leu Arg Leu Ile Lys His Thr Cys Asp

470 475 480 485

gaa ggt gct cgc ttt aac acc ccg gtc ggt gtt tgt ggt gaa gca gca 1603

Glu Gly Ala Arg Phe Asn Thr Pro Val Gly Val Cys Gly Glu Ala Ala

490 495 500

gca gac cca ctg ttg gca act gtc ctc acc ggt ctt ggc gtg aac tcc 1651

Ala Asp Pro Leu Leu Ala Thr Val Leu Thr Gly Leu Gly Val Asa Ser

505 510 515

ctg tcc gca gca tcc act gct ctc gca gca gtc ggt gca aag ctg tca 1699

Leu Ser Ala Ala Ser Thr Ala Leu Ala Ala Val Gly Ala Lys Leu Ser

520 525 530

gag gtc acc ctg gaa acc tgt aag aag gca gca gaa gca gca ctt gac 1747

Glu Val Thr Leu Glu Thr Cys Lys Lys Ala Ala Glu Ala Ala Leu Asp

535 540 545

gct gaa ggt gca act gaa gca cgc gat gct gta cgc gca gtg atc gac 1795

Ala Glu Gly Ala Thr Glu Ala Arg Asp Ala Val Arg Ala Val Ile Asp

550 555 560 565

gca gca gtc taaaccactg ttgagctaaa aag 1827

Ala Ala Val

<210>18

<211>568

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>18

Val Ala Thr Val Ala Asp Val Asn Gln Asp Thr Val Leu Lys Gly Thr

1 5 10 15

Gly Val Val Gly Gly Val Arg Tyr Ala Ser Ala Val Trp Ile Thr Pro

20 25 30

Arg Pro Glu Leu Pro Gln Ala Gly Glu Val Val Ala Glu Glu Asn Arg

35 40 45

Glu Ala Glu Gln Glu Arg Phe Asp Ala Ala Ala Ala Thr Val Ser Ser

50 55 60

Arg Leu Leu Glu Arg Ser Glu Ala Ala Glu Gly Pro Ala Ala Glu Val

65 70 75 80

Leu Lys Ala Thr Ala Gly Met Val Asn Asp Arg Gly Trp Arg Lys Ala

85 90 95

Val Ile Lys Gly Val Lys Gly Gly His Pro Ala Glu Tyr Ala Val Val

100 105 110

Ala Ala Thr Thr Lys Phe Ile Ser Met Phe Glu Ala Ala Gly Gly Leu

115 120 125

Ile Ala Glu Arg Thr Thr Asp Leu Arg Asp Ile Arg Asp Arg Val Ile

130 135 140

Ala Glu Leu Arg Gly Asp Glu Glu Pro Gly Leu Pro Ala Val Ser Gly

145 150 155 160

Gln Val Ile Leu Phe Ala Asp Asp Leu Ser Pro Ala Asp Thr Ala Ala

165 170 175

Leu Asp Thr Asp Leu Phe Val Gly Leu Val Thr Glu Leu Gly Gly Pro

180 185 190

Thr Ser His Thr Ala Ile Ile Ala Arg Gln Leu Asn Val Pro Cys Ile

195 200 205

Val Ala Ser Gly Ala Gly Ile Lys Asp Ile Lys Ser Gly Glu Lys Val

210 215 220

Leu Ile Asp Gly Ser Leu Gly Thr Ile Asp Arg Asn Ala Asp Glu Ala

225 230 235 240

Glu Ala Thr Lys Leu Val Ser Glu Ser Leu Glu Arg Ala Ala Arg Ile

245 250 255

Ala Glu Trp Lys Gly Pro Ala Gln Thr Lys Asp Gly Tyr Arg Val Gln

260 265 270

Leu Leu Ala Asn Val Gln Asp Gly Asn Ser Ala Gln Gln Ala Ala Gln

275 280 285

Thr Glu Ala Glu Gly Ile Gly Leu Phe Arg Thr Glu Leu Cys Phe Leu

290 295 300

Ser Ala Thr Glu Glu Pro Ser Val Asp Glu Gln Ala Ala Val Tyr Ser

305 310 315 320

Lys Val Leu Glu Ala Phe Pro Glu Ser Lys Val Val Val Arg Ser Leu

325 330 335

Asp Ala Gly Ser Asp Lys Pro Val Pro Phe Ala Ser Met Ala Asp Glu

340 345 350

Met Asn Pro Ala Leu Gly Val Arg Gly Leu Arg Ile Ala Arg Gly Gln

355 360 365

Val Asp Leu Leu Thr Arg Gln Leu Asp Ala Ile Ala Lys Ala Ser Glu

370 375 380

Glu Leu Gly Arg Gly Asp Asp Ala Pro Thr Trp Val Met Ala Pro Met

385 390 395 400

Val Ala Thr Ala Tyr Glu Ala Lys Trp Phe Ala Asp Met Cys Arg Glu

405 410 415

Arg Gly Leu Ile Ala Gly Ala Met Ile Glu Val Pro Ala Ala Ser Leu

420 425 430

Met Ala Asp Lys Ile Met Pro His Leu Asp Phe Val Ser Ile Gly Thr

435 440 445

Asn Asp Leu Thr Gln Tyr Thr Met Ala Ala Asp Arg Met Ser Pro Glu

450 455 460

Leu Ala Tyr Leu Thr Asp Pro Trp Gln Pro Ala Val Leu Arg Leu Ile

465 470 475 480

Lys His Thr Cys Asp Glu Gly Ala Arg Phe Asn Thr Pro Val Gly Val

485 490 495

Cys Gly Glu Ala Ala Ala Asp Pro Leu Leu Ala Thr Val Leu Thr Gly

500 505 510

Leu Gly Val Asn Ser Leu Ser Ala Ala Ser Thr Ala Leu Ala Ala Val

515 520 525

Gly Ala Lys Leu Ser Glu Val Thr Leu Glu Thr Cys Lys Lys Ala Ala

530 535 540

Glu Ala Ala Leu Asp Ala Glu Gly Ala Thr Glu Ala Arg Asp Ala Val

545 550 555 560

Arg Ala Val Ile Asp Ala Ala Val

565

<210>19

<211>1629

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(98)..(1606)

<223>FRXA01244

<400>19

agatgtcgat ttctcgagga agaagttaac gccgaagaaa accgtgaatc agagcaggag 60

cgcttcgacg ccgctgcagc cacagtctct tcttcgt ttg ctt gag cgc tcc gaa 115

Leu Leu Glu Arg Ser Glu

1 5

gct gct gaa gga cca gca gct gag gtg ctt aaa gct act gct ggc atg 163

Ala Ala Glu Gly Pro Ala Ala Glu Val Leu Lys Ala Thr Ala Gly Met

10 15 20

gtc aat gac cgt ggc tgg cgt aag gct gtc atc aag ggt gtc aag ggt 211

Val Asn Asp Arg Gly Trp Arg Lys Ala Val Ile Lys Gly Val Lys Gly

25 30 35

ggt cac cct gcg gaa tac gcc gtg gtt gca gca aca acc aag ttc atc 259

Gly His Pro Ala Glu Tyr Ala Val Val Ala Ala Thr Thr Lys Phe Ile

40 45 50

tcc atg ttc gaa gcc gca ggc ggc ctg atc gcg gag cgc acc aca gac 307

Ser Met Phe Glu Ala Ala Gly Gly Leu Ile Ala Glu Arg Thr Thr Asp

55 60 65 70

ttg cgc gac atc cgc gac cgc gtc atc gca gaa ctt cgt ggc gat gaa 355

Leu Arg Asp Ile Arg Asp Arg Val Ile Ala Glu Leu Arg Gly Asp Glu

75 80 85

gag cca ggt ctg cca gct gtt tcc gga cag gtc att ctc ttt gca gat 403

Glu Pro Gly Leu Pro Ala Val Ser Gly Gln Val Ile Leu Phe Ala Asp

90 95 100

gac ctc tcc cca gca gac acc gcg gca cta gac aca gat ctc ttt gtg 451

Asp Leu Ser Pro Ala Asp Thr Ala Ala Leu Asp Thr Asp Leu Phe Val

105 110 115

gga ctt gtc act gag ctg ggt ggc cca acg agc cac acc gcg atc atc 499

Gly Leu Val Thr Glu Leu Gly Gly Pro Thr Ser His Thr Ala Ile Ile

120 125 130

gca cgc cag ctc aac gtg cct tgc atc gtc gca tcc ggc gcc ggc atc 547

Ala Arg Gln Leu Asn Val Pro Cys Ile Val Ala Ser Gly Ala Gly Ile

135 140 145 150

aag gac atc aag tcc ggc gaa aag gtg ctt atc gac ggc agc ctc ggc 595

Lys Asp Ile Lys Ser Gly Glu Lys Val Leu Ile Asp Gly Ser Leu Gly

155 160 165

acc att gac cgc aac gcg gac gaa gct gaa gca acc aag ctc gtc tcc 643

Thr Ile Asp Arg Asn Ala Asp Glu Ala Glu Ala Thr Lys Leu Val Ser

170 175 180

gag tcc ctc gag cgc gct gct cgc atc gcc gag tgg aag ggt cct gca 691

Glu Ser Leu Glu Arg Ala Ala Arg Ile Ala Glu Trp Lys Gly Pro Ala

185 190 195

caa acc aag gac ggc tac cgc gtt cag ctg ttg gcc aac gtc caa gac 739

Gln Thr Lys Asp Gly Tyr Arg Val Gln Leu Leu Ala Asn Val Gln Asp

200 205 210

ggc aac tct gca cag cag gct gca cag acc gaa gca gaa ggc atc ggc 787

Gly Asn Ser Ala Gln Gln Ala Ala Gln Thr Glu Ala Glu Gly Ile Gly

215 220 225 230

ctg ttc cgc acc gaa ctg tgc ttc ctt tcc gcc acc gaa gag cca agc 835

Leu Phe Arg Thr Glu Leu Cys Phe Leu Ser Ala Thr Glu Glu Pro Ser

235 240 245

gtt gat gag cag gct gcg gtc tac tca aag gtg ctt gaa gca ttc cca 883

Val Asp Glu Gln Ala Ala Val Tyr Ser Lys Val Leu Glu Ala Phe Pro

250 255 260

gag tcc aag gtc gtt gtc cgc tcc ctc gac gca ggt tct gac aag cca 931

Glu Ser Lys Val Val Val Arg Ser Leu Asp Ala Gly Ser Asp Lys Pro

265 270 275

gtt cca ttc gca tcg atg gct gat gag atg aac cca gca ctg ggt gtt 979

Val Pro Phe Ala Ser Met Ala Asp Glu Met Asn Pro Ala Leu Gly Val

280 285 290

cgt ggc ctg cgt atc gca cgt gga cag gtt gat ctg ctg act cgc cag 1027

Arg Gly Leu Arg Ile Ala Arg Gly Gln Val Asp Leu Leu Thr Arg Gln

295 300 305 310

ctc gac gca att gcg aag gcc agc gaa gaa ctc ggc cgt ggc gac gac 1075

Leu Asp Ala Ile Ala Lys Ala Ser Glu Glu Leu Gly Arg Gly Asp Asp

315 320 325

gcc cca acc tgg gtt atg gct cca atg gtg gct acc gct tat gaa gca 1123

Ala Pro Thr Trp Val Met Ala Pro Met Val Ala Thr Ala Tyr Glu Ala

330 335 340

aag tgg ttt gct gac atg tgc cgt gag cgt ggc cta atc gcc ggc gcc 1171

Lys Trp Phe Ala Asp Met Cys Arg Glu Arg Gly Leu Ile Ala Gly Ala

345 350 355

atg atc gaa gtt cca gca gca tcc ctg atg gca gac aag atc atg cct 1219

Met Ile Glu Val Pro Ala Ala Ser Leu Met Ala Asp Lys Ile Met Pro

360 365 370

cac ctg gac ttt gtt tcc atc ggt acc aac gac ctg acc cag tac acc 1267

His Leu Asp Phe Val Ser Ile Gly Thr Asn Asp Leu Thr Gln Tyr Thr

375 380 385 390

atg gca gcg gac cgc atg tct cct gag ctt gcc tac ctg acc gat cct 1315

Met Ala Ala Asp Arg Met Ser Pro Glu Leu Ala Tyr Leu Thr Asp Pro

395 400 405

tgg cag cca gca gtc ctg cgc ctg atc aag cac acc tgt gac gaa ggt 1363

Trp Gln Pro Ala Val Leu Arg Leu Ile Lys His Thr Cys Asp Glu Gly

410 415 420

gct cgc ttt aac acc ccg gtc ggt gtt tgt ggt gaa gca gca gca gac 1411

Ala Arg Phe Asn Thr Pro Val Gly Val Cys Gly Glu Ala Ala Ala Asp

425 430 435

cca ctg ttg gca act gtc ctc acc ggt ctt ggc gtg aac tcc ctg tcc 1459

Pro Leu Leu Ala Thr Val Leu Thr Gly Leu Gly Val Asn Ser Leu Ser

440 445 450

gca gca tcc act gct ctc gca gca gtc ggt gca aag ctg tca gag gtc 1507

Ala Ala Ser Thr Ala Leu Ala Ala Val Gly Ala Lys Leu Ser Glu Val

455 460 465 470

acc ctg gaa acc tgt aag aag gca gca gaa gca gca ctt gac gct gaa 1555

Thr Leu Glu Thr Cys Lys Lys Ala Ala Glu Ala Ala Leu Asp Ala Glu

475 480 485

ggt gca act gaa gca cgc gat gct gta cgc gca gtg atc gac gca gca 1603

Gly Ala Thr Glu Ala Arg Asp Ala Val Arg Ala Val Ile Asp Ala Ala

490 495 500

gtc taaaccactg ttgagctaaa aag 1629

Val

<210>20

<211>503

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>20

Leu Leu Glu Arg Ser Glu Ala Ala Glu Gly Pro Ala Ala Glu Val Leu

1 5 10 15

Lys Ala Thr Ala Gly Met Val Asn Asp Arg Gly Trp Arg Lys Ala Val

20 25 30

Ile Lys Gly Val Lys Gly Gly His Pro Ala Glu Tyr Ala Val Val Ala

35 40 45

Ala Thr Thr Lys Phe Ile Ser Met Phe Glu Ala Ala Gly Gly Leu Ile

50 55 60

Ala Glu Arg Thr Thr Asp Leu Arg Asp Ile Arg Asp Arg Val Ile Ala

65 70 75 80

Glu Leu Arg Gly Asp Glu Glu Pro Gly Leu Pro Ala Val Ser Gly Gln

85 90 95

Val Ile Leu Phe Ala Asp Asp Leu Ser Pro Ala Asp Thr Ala Ala Leu

100 105 110

Asp Thr Asp Leu Phe Val Gly Leu Val Thr Glu Leu Gly Gly Pro Thr

115 120 125

Ser His Thr Ala Ile Ile Ala Arg Gln Leu Asn Val Pro Cys Ile Val

130 135 140

Ala Ser Gly Ala Gly Ile Lys Asp Ile Lys Ser Gly Glu Lys Val Leu

145 150 155 160

Ile Asp Gly Ser Leu Gly Thr Ile Asp Arg Asn Ala Asp Glu Ala Glu

165 170 175

Ala Thr Lys Leu Val Ser Glu Ser Leu Glu Arg Ala Ala Arg Ile Ala

180 185 190

Glu Trp Lys Gly Pro Ala Gln Thr Lys Asp Gly Tyr Arg Val Gln Leu

195 200 205

Leu Ala Asn Val Gln Asp Gly Asn Ser Ala Gln Gln Ala Ala Gln Thr

210 215 220

Glu Ala Glu Gly Ile Gly Leu Phe Arg Thr Glu Leu Cys Phe Leu Ser

225 230 235 240

Ala Thr Glu Glu Pro Ser Val Asp Glu Gln Ala Ala Val Tyr Ser Lys

245 250 255

Val Leu Glu Ala Phe Pro Glu Ser Lys Val Val Val Arg Ser Leu Asp

260 265 270

Ala Gly Ser Asp Lys Pro Val Pro Phe Ala Ser Met Ala Asp Glu Met

275 280 285

Asn Pro Ala Leu Gly Val Arg Gly Leu Arg Ile Ala Arg Gly Gln Val

290 295 300

Asp Leu Leu Thr Arg Gln Leu Asp Ala Ile Ala Lys Ala Ser Glu Glu

305 310 315 320

Leu Gly Arg Gly Asp Asp Ala Pro Thr Trp Val Met Ala Pro Met Val

325 330 335

Ala Thr Ala Tyr Glu Ala Lys Trp Phe Ala Asp Met Cys Arg Glu Arg

340 345 350

Gly Leu Ile Ala Gly Ala Met Ile Glu Val Pro Ala Ala Ser Leu Met

355 360 365

Ala Asp Lys Ile Met Pro His Leu Asp Phe Val Ser Ile Gly Thr Asn

370 375 380

Asp Leu Thr Gln Tyr Thr Met Ala Ala Asp Arg Met Ser Pro Glu Leu

385 390 395 400

Ala Tyr Leu Thr Asp Pro Trp Gln Pro Ala Val Leu Arg Leu Ile Lys

405 410 415

His Thr Cys Asp Glu Gly Ala Arg Phe Asn Thr Pro Val Gly Val Cys

420 425 430

Gly Glu Ala Ala Ala Asp Pro Leu Leu Ala Thr Val Leu Thr Gly Leu

435 440 445

Gly Val Asn Ser Leu Ser Ala Ala Ser Thr Ala Leu Ala Ala Val Gly

450 455 460

Ala Lys Leu Ser Glu Val Thr Leu Glu Thr Cys Lys Lys Ala Ala Glu

465 470 475 480

Ala Ala Leu Asp Ala Glu Gly Ala Thr Glu Ala Arg Asp Ala Val Arg

485 490 495

Ala Val Ile Asp Ala Ala Val

500

<210>21

<211>390

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(367)

<223>RXA01300

<400>21

gatcgacatt aaatcccctc ccttgggggg tttaactaac aaatcgctgc gccctaatcc 60

gttcggatta acggcgtagc aacacgaaag gacactttcc atg gct tcc aag act 115

Met Ala Ser Lys Thr

1 5

gta acc gtc ggt tcc tcc gtt ggc ctg cac gca cgt cca gca tcc atc 163

Val Thr Val Gly Ser Ser Val Gly Leu His Ala Arg Pro Ala Ser Ile

10 15 20

atc gct gaa gcg gct gct gag tac gac gac gaa atc ttg ctg acc ctg 211

Ile Ala Glu Ala Ala Ala Glu Tyr Asp Asp Glu Ile Leu Leu Thr Leu

25 30 35

gtt ggc tcc gat gat gac gaa gag acc gac gcg tcc tct tcc ctc atg 259

Val Gly Ser Asp Asp Asp Glu Glu Thr Asp Ala Ser Ser Ser Leu Met

40 45 50

atc atg gcg ctg ggc gca gag cac ggc aac gaa gtt acc gtc acc tcc 307

Ile Met Ala Leu Gly Ala Glu His Gly Asn Glu Val Thr Val Thr Ser

55 60 65

gac aac gct gaa gct gtt gag aag atc gct gcg ctt atc gca cag gac 355

Asp Asn Ala Glu Ala Val Glu Lys Ile Ala Ala Leu Ile Ala Gln Asp

70 75 80 85

ctt gac gct gag taaacaacgc tctgcttgtt aaa 390

Leu Asp Ala Glu

<210>22

<211>89

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>22

Met Ala Ser Lys Thr Val Thr Val Gly Ser Ser Val Gly Leu His Ala

1 5 10 15

Arg Pro Ala Ser Ile Ile Ala Glu Ala Ala Ala Glu Tyr Asp Asp Glu

20 25 30

Ile Leu Leu Thr Leu Val Gly Ser Asp Asp Asp Glu Glu Thr Asp Ala

35 40 45

Ser Ser Ser Leu Met Ile Met Ala Leu Gly Ala Glu His Gly Asn Glu

50 55 60

Val Thr Val Thr Ser Asp Asn Ala Glu Ala Val Glu Lys Ile Ala Ala

65 70 75 80

Leu Ile Ala Gln Asp Leu Asp Ala Glu

85

<210>23

<211>508

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(508)

<223>RXN03002

<400>23

ggaacttcga ggtgtcttcg tggggcgtac ggagatctag caagtgtggc tttatgtttg 60

accctatccg aatcaacatg cagtgaatta acatctactt atg ttt gta ctc aaa 115

Met Phe Val Leu Lys

1 5

gat ctg cta aag gca gaa cgc ata gaa ctc gac cgc acg gtc acc gat 163

Asp Leu Leu Lys Ala Glu Arg Ile Glu Leu Asp Arg Thr Val Thr Asp

10 15 20

tgg cgt gaa ggc atc cgc gcc gca ggt gta ctc cta gaa aag aca aac 211

Trp Arg Glu Gly Ile Arg Ala Ala Gly Val Leu Leu Glu Lys Thr Asn

25 30 35

agc att gat tcc gcc tac acc gat gcc atg atc gcc agc gtg gaa gaa 259

Ser Ile Asp Ser Ala Tyr Thr Asp Ala Met Ile Ala Ser Val Glu Glu

40 45 50

aaa ggc ccc tac att gtg gtc gct cca ggt ttc gct ttc gcg cac gcc 307

Lys Gly Pro Tyr Ile Val Val Ala Pro Gly Phe Ala Phe Ala His Ala

55 60 65

cgc ccc agc aga gca gtc cgc gag acc gct atg tcg tgg gtg cgc ctg 355

Arg Pro Ser Arg Ala Val Arg Glu Thr Ala Met Ser Trp Val Arg Leu

70 75 80 85

gcc tcc cct gtt tcc ttc ggt cac agt aag aat gat ccc ctc aat ctc 403

Ala Ser Pro Val Ser Phe Gly His Ser Lys Asn Asp Pro Leu Asn Leu

90 95 100

atc gtt gct ctc gct gcc aaa gat gcc acc gca cat acc caa gcg atg 451

Ile Val Ala Leu Ala Ala Lys Asp Ala Thr Ala His Thr Gln Ala Met

105 110 115

gcg gca ttg gct aaa gct tta gga aaa tac cga aag gat ctc gac gag 499

Ala Ala Leu Ala Lys Ala Leu Gly Lys Tyr Arg Lys Asp Leu Asp Glu

120 125 130

gca caa agt 508

Ala Gln Ser

135

<210>24

<211>136

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>24

Met Phe Val Leu Lys Asp Leu Leu Lys Ala Glu Arg Ile Glu Leu Asp

1 5 10 15

Arg Thr Val Thr Asp Trp Arg Glu Gly Ile Arg Ala Ala Gly Val Leu

20 25 30

Leu Glu Lys Thr Asn Ser Ile Asp Ser Ala Tyr Thr Asp Ala Met Ile

35 40 45

Ala Ser Val Glu Glu Lys Gly Pro Tyr Ile Val Val Ala Pro Gly Phe

50 55 60

Ala Phe Ala His Ala Arg Pro Ser Arg Ala Val Arg Glu Thr Ala Met

65 70 75 80

Ser Trp Val Arg Leu Ala Ser Pro Val Ser Phe Gly His Ser Lys Asn

85 90 95

Asp Pro Leu Asn Leu Ile Val Ala Leu Ala Ala Lys Asp Ala Thr Ala

100 105 110

His Thr Gln Ala Met Ala Ala Leu Ala Lys Ala Leu Gly Lys Tyr Arg

115 120 125

Lys Asp Leu Asp Glu Ala Gln Ser

130 135

<210>25

<211>789

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(14)..(766)

<223>RXC00953

<400>25

cttgcattcc cca atg gcg cca cca acg gta ggc aac tac atc atg cag tcc 52

Met Ala Pro Pro Thr Val Gly Asn Tyr Ile Met Gln Ser

1 5 10

ttc act caa ggt ctg cag ttc ggc gtt gca gtt gcc gtg att ctc ttt 100

Phe Thr Gln Gly Leu Gln Phe Gly Val Ala Val Ala Val Ile Leu Phe

15 20 25

ggt gtc cgc acc att ctt ggt gaa ctg gtc ccc gca ttc caa ggt att 148

Gly Val Arg Thr Ile Leu Gly Glu Leu Val Pro Ala Phe Gln Gly Ile

30 35 40 45

gct gcg aag gtt gtt ccc gga gct atc ccc gca ttg gat gca ccg atc 196

Ala Ala Lys Val Val Pro Gly Ala Ile Pro Ala Leu Asp Ala Pro Ile

50 55 60

gtg ttc ccc tac gcg cag aac gcc gtt ctc att ggt ttc ttg tct tcc 244

Val Phe Pro Tyr Ala Gln Asn Ala Val Leu Ile Gly Phe Leu Ser Ser

65 70 75

ttc gtc ggt ggc ttg gtt ggc ctg act gtt ctt gca tcg tgg ctg aac 292

Phe Val Gly Gly Leu Val Gly Leu Thr Val Leu Ala Ser Trp Leu Asn

80 85 90

cca gct ttt ggt gtc gcg ttg att ctg cct ggt ttg gtc ccc cac ttc 340

Pro Ala Phe Gly Val Ala Leu Ile Leu Pro Gly Leu Val Pro His Phe

95 100 105

ttc act ggt ggc gcg gcg ggc gtt tac ggt aat gcc acg ggt ggt cgt 388

Phe Thr Gly Gly Ala Ala Gly Val Tyr Gly Asn Ala Thr Gly Gly Arg

110 115 120 125

cga gga gca gta ttt ggc gcc ttt gcc aac ggt ctt ctg att acc ttc 436

Arg Gly Ala Val Phe Gly Ala Phe Ala Asn Gly Leu Leu Ile Thr Phe

130 135 140

ctc cct gct ttc ctg ctt ggt gtg ctt ggt tcc ttc ggg tca gag aac 484

Leu Pro Ala Phe Leu Leu Gly Val Leu Gly Ser Phe Gly Ser Glu Asn

145 150 155

acc act ttc ggt gat gcg gac ttt ggt tgg ttc gga atc gtt gtt ggt 532

Thr Thr Phe Gly Asp Ala Asp Phe Gly Trp Phe Gly Ile Val Val Gly

160 165 170

tct gca gcc aag gtg gaa ggt gct ggc ggg ctc atc ttg ttg ctc atc 580

Ser Ala Ala Lys Val Glu Gly Ala Gly Gly Leu Ile Leu Leu Leu Ile

175 180 185

atc gca gcg gtt ctt ctg ggt ggc gcg atg gtc ttc cag aag cgc gtc 628

Ile Ala Ala Val Leu Leu Gly Gly Ala Met Val Phe Gln Lys Arg Val

190 195 200 205

gtg aat ggg cac tgg gat cca gct ccc aac cgt gag cgc gtg gag aag 676

Val Asn Gly His Trp Asp Pro Ala Pro Asn Arg Glu Arg Val Glu Lys

210 215 220

gcg gaa gct gat gcc act cca acg gct ggg gct cgg acc tac cct aag 724

Ala Glu Ala Asp Ala Thr Pro Thr Ala Gly Ala Arg Thr Tyr Pro Lys

225 230 235

att gct cct ccg gcg ggc gct cct acc cca ccg gct cga agc 766

Ile Ala Pro Pro Ala Gly Ala Pro Thr Pro Pro Ala Arg Ser

240 245 250

taagatctcc aaaaccctga gat 789

<210>26

<211>251

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>26

Met Ala Pro Pro Thr Val Gly Asn Tyr Ile Met Gln Ser Phe Thr Gln

1 5 10 15

Gly Leu Gln Phe Gly Val Ala Val Ala Val Ile Leu Phe Gly Val Arg

20 25 30

Thr Ile Leu Gly Glu Leu Val Pro Ala Phe Gln Gly Ile Ala Ala Lys

35 40 45

Val Val Pro Gly Ala Ile Pro Ala Leu Asp Ala Pro Ile Val Phe Pro

50 55 60

Tyr Ala Gln Asn Ala Val Leu Ile Gly Phe Leu Ser Ser Phe Val Gly

65 70 75 80

Gly Leu Val Gly Leu Thr Val Leu Ala Ser Trp Leu Asn Pro Ala Phe

85 90 95

Gly Val Ala Leu Ile Leu Pro Gly Leu Val Pro His Phe Phe Thr Gly

100 105 110

Gly Ala Ala Gly Val Tyr Gly Asn Ala Thr Gly Gly Arg Arg Gly Ala

115 120 125

Val Phe Gly Ala Phe Ala Asn Gly Leu Leu Ile Thr Phe Leu Pro Ala

130 135 140

Phe Leu Leu Gly Val Leu Gly Ser Phe Gly Ser Glu Asn Thr Thr Phe

145 150 155 160

Gly Asp Ala Asp Phe Gly Trp Phe Gly Ile Val Val Gly Ser Ala Ala

165 170 175

Lys Val Glu Gly Ala Gly Gly Leu Ile Leu Leu Leu Ile Ile Ala Ala

180 185 190

Val Leu Leu Gly Gly Ala Met Val Phe Gln Lys Arg Val Val Asn Gly

195 200 205

His Trp Asp Pro Ala Pro Asn Arg Glu Arg Val Glu Lys Ala Glu Ala

210 215 220

Asp Ala Thr Pro Thr Ala Gly Ala Arg Thr Tyr Pro Lys Ile Ala Pro

225 230 235 240

Pro Ala Gly Ala Pro Thr Pro Pro Ala Arg Ser

245 250

<210>27

<211>553

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(553)

<223>RXC03001

<400>27

cccggttcac gtgatcaatg acttcacgag caccgatgaa atcgatgctg cgcttcgtga 60

acgctacgac atctaactac tttaaaagga cgaaaatatt atg gac tgg tta acc 115

Met Asp Trp Leu Thr

1 5

att cct ctt ttc ctc gtt aat gaa atc ctt gcg gtt ccg gct ttc ctc 163

Ile Pro Leu Phe Leu Val Asn Glu Ile Leu Ala Val Pro Ala Phe Leu

10 15 20

atc ggt atc atc acc gcc gtg gga ttg ggt gcc atg ggg cgt tcc gtc 211

Ile Gly Ile Ile Thr Ala Val Gly Leu Gly Ala Met Gly Arg Ser Val

25 30 35

ggt cag gtt atc ggt gga gca atc aaa gca acg ttg ggc ttt ttg ctc 259

Gly Gln Val Ile Gly Gly Ala Ile Lys Ala Thr Leu Gly Phe Leu Leu

40 45 50

att ggt gcg ggt gcc acg ttg gtc act gcc tcc ctg gag cca ctg ggt 307

Ile Gly Ala Gly Ala Thr Leu Val Thr Ala Ser Leu Glu Pro Leu Gly

55 60 65

gcg atg atc atg ggt gcc aca ggc atg cgt ggt gtt gtc cca acg aat 355

Ala Met Ile Met Gly Ala Thr Gly Met Arg Gly Val Val Pro Thr Asn

70 75 80 85

gaa gcc atc gcc gga atc gca cag gct gaa tac ggc gcg cag gtg gcg 403

Glu Ala Ile Ala Gly Ile Ala Gln Ala Glu Tyr Gly Ala Gln Val Ala

90 95 100

tgg ctg atg att ctg ggc ttc gcc atc tct ttg gtg ttg gct cgt ttc 451

Trp Leu Met Ile Leu Gly Phe Ala Ile Ser Leu Val Leu Ala Arg Phe

105 110 115

acc aac ctg cgt tat gtc ttg ctc aac gga cac cac gtg ctg ttg atg 499

Thr Asn Leu Arg Tyr Val Leu Leu Asn Gly His His Val Leu Leu Met

120 125 130

tgc acc atg ctc acc atg gtc ttg gcc acc gga aga gtt gat gcg tgg 547

Cys Thr Met Leu Thr Met Val Leu Ala Thr Gly Arg Val Asp Ala Trp

135 140 145

atc ttc 553

Ile Phe

150

<210>28

<211>151

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>28

Met Asp Trp Leu Thr Ile Pro Leu Phe Leu Val Asn Glu Ile Leu Ala

1 5 10 15

Val Pro Ala Phe Leu Ile Gly Ile Ile Thr Ala Val Gly Leu Gly Ala

20 25 30

Met Gly Arg Ser Val Gly Gln Val Ile Gly Gly Ala Ile Lys Ala Thr

35 40 45

Leu Gly Phe Leu Leu Ile Gly Ala Gly Ala Thr Leu Val Thr Ala Ser

50 55 60

Leu Glu Pro Leu Gly Ala Met Ile Met Gly Ala Thr Gly Met Arg Gly

65 70 75 80

Val Val Pro Thr Asn Glu Ala Ile Ala Gly Ile Ala Gln Ala Glu Tyr

85 90 95

Gly Ala Gln Val Ala Trp Leu Met Ile Leu Gly Phe Ala Ile Ser Leu

100 105 110

Val Leu Ala Arg Phe Thr Asn Leu Arg Tyr Val Leu Leu Asn Gly His

115 120 125

His Val Leu Leu Met Cys Thr Met Leu Thr Met Val Leu Ala Thr Gly

130 135 140

Arg Val Asp Ala Trp Ile Phe

145 150

<210>29

<211>2172

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(2149)

<223>RXN01943

<400>29

ccgattcttt ttcggcccaa ttcgtaacgg cgatcctctt aagtggacaa gaaagtctct 60

tgcccgcggg agacagaccc tacgtttaga aaggtttgac atg gcg tcc aaa ctg 115

Met Ala Ser Lys Leu

1 5

acg acg aca tcg caa cat att ctg gaa aac ctt ggt gga cca gac aat 163

Thr Thr Thr Ser Gln His Ile Leu Glu Asn Leu Gly Gly Pro Asp Asn

10 15 20

att act tcg atg act cac tgt gcg act cgc ctt cgc ttc caa gtg aag 211

Ile Thr Ser Met Thr His Cys Ala Thr Arg Leu Arg Phe Gln Val Lys

25 30 35

gat caa tcc att gtt gat caa caa gaa att gac tcc gac cca tca gtt 259

Asp Gln Ser Ile Val Asp Gln Gln Glu Ile Asp Ser Asp Pro Ser Val

40 45 50

ctt ggc gta gta ccc caa gga tcc acc ggt atg cag gtg gtg atg ggt 307

Leu Gly Val Val Pro Gln Gly Ser Thr Gly Met Gln Val Val Met Gly

55 60 65

gga tct gtt gca aac tat tac caa gaa atc ctc aaa ctt gat gga atg 355

Gly Ser Val Ala Asn Tyr Tyr Gln Glu Ile Leu Lys Leu Asp Gly Met

70 75 80 85

aag cac ttc gcc gac ggt gaa gct aca gag agt tca tcc aag aag gaa 403

Lys His Phe Ala Asp Gly Glu Ala Thr Glu Ser Ser Ser Lys Lys Glu

90 95 100

tac ggc gga gtc cgt ggc aag tac tcg tgg att gac tac gcc ttc gag 451

Tyr Gly Gly Val Arg Gly Lys Tyr Ser Trp Ile Asp Tyr Ala Phe Glu

105 110 115

ttc ttg tct gat act ttc cga cca atc ctg tgg gcc ctg ctt ggt gcc 499

Phe Leu Ser Asp Thr Phe Arg Pro Ile Leu Trp Ala Leu Leu Gly Ala

120 125 130

tca ctg att att acc ttg ttg gtt ctt gcg gat act ttc ggt ttg caa 547

Ser Leu Ile Ile Thr Leu Leu Val Leu Ala Asp Thr Phe Gly Leu Gln

135 140 145

gac ttc cgc gct cca atg gat gag cag cct gat act tat gta ttc ctg 595

Asp Phe Arg Ala Pro Met Asp Glu Gln Pro Asp Thr Tyr Val Phe Leu

150 155 160 165

cac tcc atg tgg cgc tcg gtc ttc tac ttc ctg cca att atg gtt ggt 643

His Ser Met Trp Arg Ser Val Phe Tyr Phe Leu Pro Ile Met Val Gly

170 175 180

gcc acc gca gct cga aag ctc ggc gca aac gag tgg att ggt gca gct 691

Ala Thr Ala Ala Arg Lys Leu Gly Ala Asn Glu Trp Ile Gly Ala Ala

185 190 195

att cca gcc gca ctt ctt act cca gaa ttc ttg gca ctg ggt tct gcc 739

Ile Pro Ala Ala Leu Leu Thr Pro Glu Phe Leu Ala Leu Gly Ser Ala

200 205 210

ggc gat acc gtc aca gtc ttt ggc ctg cca atg gtt ctg aat gac tac 787

Gly Asp Thr Val Thr Val Phe Gly Leu Pro Met Val Leu Asn Asp Tyr

215 220 225

tcc gga cag gta ttc cca ccg ctg att gca gca att ggt ctg tac tgg 835

Ser Gly Gln Val Phe Pro Pro Leu Ile Ala Ala Ile Gly Leu Tyr Trp

230 235 240 245

gtg gaa aag gga ctg aag aag atc atc cct gaa gca gtc caa atg gtg 883

Val Glu Lys Gly Leu Lys Lys Ile Ile Pro Glu Ala Val Gln Met Val

250 255 260

ttc gtc cca ttc ttc tcc ctg ctg att atg atc cca gcg acc gca ttc 931

Phe Val Pro Phe Phe Ser Leu Leu Ile Met Ile Pro Ala Thr Ala Phe

265 270 275

ctg ctt gga cct ttc ggc atc ggt gtt ggt aac gga att tcc aac ctg 979

Leu Leu Gly Pro Phe Gly Ile Gly Val Gly Asn Gly Ile Ser Asn Leu

280 285 290

ctt gaa gcg att aac aac ttc agc cca ttt att ctt tcc atc gtt atc 1027

Leu Glu Ala Ile Asn Asn Phe Ser Pro Phe Ile Leu Ser Ile Val Ile

295 300 305

cca ttg ctc tac cca ttc ttg gtt cca ctt gga ttg cac tgg cca cta 1075

Pro Leu Leu Tyr Pro Phe Leu Val Pro Leu Gly Leu His Trp Pro Leu

310 315 320 325

aac gcc atc atg atc cag aac atc aac acc ctg ggt tac gac ttc att 1123

Asn Ala Ile Met Ile Gln Asn Ile Asn Thr Leu Gly Tyr Asp Phe Ile

330 335 340

cag gga cca atg ggt gcc tgg aac ttc gcc tgc ttc ggc ctg gtc acc 1171

Gln Gly Pro Met Gly Ala Trp Asn Phe Ala Cys Phe Gly Leu Val Thr

345 350 355

ggc gtg ttc ttg ctc tcc att aag gaa cga aac aag gcc atg cgt cag 1219

Gly Val Phe Leu Leu Ser Ile Lys Glu Arg Asn Lys Ala Met Arg Gln

360 365 370

gtt tcc ctg ggt ggc atg ttg gct ggt ttg ctc ggc ggc att tcc gag 1267

Val Ser Leu Gly Gly Met Leu Ala Gly Leu Leu Gly Gly Ile Ser Glu

375 380 385

cct tcc ctc tac ggt gtt ctg ctc cga ttc aag aag acc tac ttc cgc 1315

Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys Lys Thr Tyr Phe Arg

390 395 400 405

ctc ctg ccg ggt tgt ttg gca ggc ggt atc gtg atg ggc atc ttc gac 1363

Leu Leu Pro Gly Cys Leu Ala Gly Gly Ile Val Met Gly Ile Phe Asp

410 415 420

atc aag gcg tac gct ttc gtg ttc acc tcc ttg ctt acc atc cca gca 1411

Ile Lys Ala Tyr Ala Phe Val Phe Thr Ser Leu Leu Thr Ile Pro Ala

425 430 435

atg gac cca tgg ttg ggc tac acc att ggt atc gca gtt gca ttc ttc 1459

Met Asp Pro Trp Leu Gly Tyr Thr Ile Gly Ile Ala Val Ala Phe Phe

440 445 450

gtt tcc atg ttc ctt gtt ctc gca ctg gac tac cgt tcc aac gaa gag 1507

Val Ser Met Phe Leu Val Leu Ala Leu Asp Tyr Arg Ser Asn Glu Glu

455 460 465

cgc gat gag gca cgt gca aag gtt gct gct gac aag cag gca gaa gaa 1555

Arg Asp Glu Ala Arg Ala Lys Val Ala Ala Asp Lys Gln Ala Glu Glu

470 475 480 485

gat ctg aag gca gaa gct aat gca act cct gca gct cca gta gct gct 1603

Asp Leu Lys Ala Glu Ala Asn Ala Thr Pro Ala Ala Pro Val Ala Ala

490 495 500

gca ggt gcg gga gcc ggt gca ggt gca gga gcc gct gct ggc gct gca 1651

Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Ala

505 510 515

acc gcc gtg gca gct aag ccg aag ctg gcc gct ggg gaa gta gtg gac 1699

Thr Ala Val Ala Ala Lys Pro Lys Leu Ala Ala Gly Glu Val Val Asp

520 525 530

att gtt tcc cca ctc gaa ggc aag gca att cca ctt tct gaa gta cct 1747

Ile Val Ser Pro Leu Glu Gly Lys Ala Ile Pro Leu Ser Glu Val Pro

535 540 545

gac cca atc ttt gca gca ggc aag ctt gga cca ggc att gca atc caa 1795

Asp Pro Ile Phe Ala Ala Gly Lys Leu Gly Pro Gly Ile Ala Ile Gln

550 555 560 565

cca act gga aac acc gtt gtt gct cca gca gac gct act gtc atc ctt 1843

Pro Thr Gly Asn Thr Val Val Ala Pro Ala Asp Ala Thr Val Ile Leu

570 575 580

gtc cag aaa tct gga cac gca gtg gca ttg cgc tta gat agc gga gtt 1891

Val Gln Lys Ser Gly His Ala Val Ala Leu Arg Leu Asp Ser Gly Val

585 590 595

gaa atc ctt gtc cac gtt gga ttg gac acc gtg caa ttg ggc ggc gaa 1939

Glu Ile Leu Val His Val Gly Leu Asp Thr Val Gln Leu Gly Gly Glu

600 605 610

ggc ttc acc gtt cac gtt gag cgc agg cag caa gtc aag gcg ggg gat 1987

Gly Phe Thr Val His Val Glu Arg Arg Gln Gln Val Lys Ala Gly Asp

615 620 625

cca ctg atc act ttt gac gct gac ttc att cga tcc aag gat cta cct 2035

Pro Leu Ile Thr Phe Asp Ala Asp Phe Ile Arg Ser Lys Asp Leu Pro

630 635 640 645

ttg atc acc cca gtt gtg gtg tct aac gcc gcg aaa ttc ggt gaa att 2083

Leu Ile Thr Pro Val Val Val Ser Asn Ala Ala Lys Phe Gly Glu Ile

650 655 660

gaa ggt att cct gca gat cag gca aat tct tcc acg act gtg atc aag 2131

Glu Gly Ile Pro Ala Asp Gln Ala Asn Ser Ser Thr Thr Val Ile Lys

665 670 675

gtc aac ggc aag aac gag taacctggga tccatgttgc gca 2172

Val Asn Gly Lys Asn Glu

680

<210>30

<211>683

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>30

Met Ala Ser Lys Leu Thr Thr Thr Ser Gln His Ile Leu Glu Asn Leu

1 5 10 15

Gly Gly Pro Asp Asn Ile Thr Ser Met Thr His Cys Ala Thr Arg Leu

20 25 30

Arg Phe Gln Val Lys Asp Gln Ser Ile Val Asp Gln Gln Glu Ile Asp

35 40 45

Ser Asp Pro Ser Val Leu Gly Val Val Pro Gln Gly Ser Thr Gly Met

50 55 60

Gln Val Val Met Gly Gly Ser Val Ala Asn Tyr Tyr Gln Glu Ile Leu

65 70 75 80

Lys Leu Asp Gly Met Lys His Phe Ala Asp Gly Glu Ala Thr Glu Ser

85 90 95

Ser Ser Lys Lys Glu Tyr Gly Gly Val Arg Gly Lys Tyr Ser Trp Ile

100 105 110

Asp Tyr Ala Phe Glu Phe Leu Ser Asp Thr Phe Arg Pro Ile Leu Trp

115 120 125

Ala Leu Leu Gly Ala Ser Leu Ile Ile Thr Leu Leu Val Leu Ala Asp

130 135 140

Thr Phe Gly Leu Gln Asp Phe Arg Ala Pro Met Asp Glu Gln Pro Asp

145 150 155 160

Thr Tyr Val Phe Leu His Ser Met Trp Arg Ser Val Phe Tyr Phe Leu

165 170 175

Pro Ile Met Val Gly Ala Thr Ala Ala Arg Lys Leu Gly Ala Asn Glu

180 185 190

Trp Ile Gly Ala Ala Ile Pro Ala Ala Leu Leu Thr Pro Glu Phe Leu

195 200 205

Ala Leu Gly Ser Ala Gly Asp Thr Val Thr Val Phe Gly Leu Pro Met

210 215 220

Val Leu Asn Asp Tyr Ser Gly Gln Val Phe Pro Pro Leu Ile Ala Ala

225 230 235 240

Ile Gly Leu Tyr Trp Val Glu Lys Gly Leu Lys Lys Ile Ile Pro Glu

245 250 255

Ala Val Gln Met Val Phe Val Pro Phe Phe Ser Leu Leu Ile Met Ile

260 265 270

Pro Ala Thr Ala Phe Leu Leu Gly Pro Phe Gly Ile Gly Val Gly Asn

275 280 285

Gly Ile Ser Asn Leu Leu Glu Ala Ile Asn Asn Phe Ser Pro Phe Ile

290 295 300

Leu Ser Ile Val Ile Pro Leu Leu Tyr Pro Phe Leu Val Pro Leu Gly

305 310 315 320

Leu His Trp Pro Leu Asn Ala Ile Met Ile Gln Asn Ile Asn Thr Leu

325 330 335

Gly Tyr Asp Phe Ile Gln Gly Pro Met Gly Ala Trp Asn Phe Ala Cys

340 345 350

Phe Gly Leu Val Thr Gly Val Phe Leu Leu Ser Ile Lys Glu Arg Asn

355 360 365

Lys Ala Met Arg Gln Val Ser Leu Gly Gly Met Leu Ala Gly Leu Leu

370 375 380

Gly Gly Ile Ser Glu Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys

385 390 395 400

Lys Thr Tyr Phe Arg Leu Leu Pro Gly Cys Leu Ala Gly Gly Ile Val

405 410 415

Met Gly Ile Phe Asp Ile Lys Ala Tyr Ala Phe Val Phe Thr Ser Leu

420 425 430

Leu Thr Ile Pro Ala Met Asp Pro Trp Leu Gly Tyr Thr Ile Gly Ile

435 440 445

Ala Val Ala Phe Phe Val Ser Met Phe Leu Val Leu Ala Leu Asp Tyr

450 455 460

Arg Ser Asn Glu Glu Arg Asp Glu Ala Arg Ala Lys Val Ala Ala Asp

465 470 475 480

Lys Gln Ala Glu Glu Asp Leu Lys Ala Glu Ala Asn Ala Thr Pro Ala

485 490 495

Ala Pro Val Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

500 505 510

Ala Ala Gly Ala Ala Thr Ala Val Ala Ala Lys Pro Lys Leu Ala Ala

515 520 525

Gly Glu Val Val Asp Ile Val Ser Pro Leu Glu Gly Lys Ala Ile Pro

530 535 540

Leu Ser Glu Val Pro Asp Pro Ile Phe Ala Ala Gly Lys Leu Gly Pro

545 550 555 560

Gly Ile Ala Ile Gln Pro Thr Gly Asn Thr Val Val Ala Pro Ala Asp

565 570 575

Ala Thr Val Ile Leu Val Gln Lys Ser Gly His Ala Val Ala Leu Arg

580 585 590

Leu Asp Ser Gly Val Glu Ile Leu Val His Val Gly Leu Asp Thr Val

595 600 605

Gln Leu Gly Gly Glu Gly Phe Thr Val His Val Glu Arg Arg Gln Gln

610 615 620

Val Lys Ala Gly Asp Pro Leu Ile Thr Phe Asp Ala Asp Phe Ile Arg

625 630 635 640

Ser Lys Asp Leu Pro Leu Ile Thr Pro Val Val Val Ser Asn Ala Ala

645 650 655

Lys Phe Gly Glu Ile Glu Gly Ile Pro Ala Asp Gln Ala Asn Ser Ser

660 665 670

Thr Thr Val Ile Lys Val Asn Gly Lys Asn Glu

675 680

<210>31

<211>1339

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(1339)

<223>FRXA02191

<400>31

ccgattcttt ttcggcccaa ttcgtaacgg cgatcctctt aagtggacaa gaaagtctct 60

tgcccgcggg agacagaccc tacgtttaga aaggtttgac atg gcg tcc aaa ctg 115

Met Ala Ser Lys Leu

1 5

acg acg aca tcg caa cat att ctg gaa aac ctt ggt gga cca gac aat 163

Thr Thr Thr Ser Gln His Ile Leu Glu Asn Leu Gly Gly Pro Asp Asn

10 15 20

att act tcg atg act cac tgt gcg act cgc ctt cgc ttc caa gtg aag 211

Ile Thr Ser Met Thr His Cys Ala Thr Arg Leu Arg Phe Gln Val Lys

25 30 35

gat caa tcc att gtt gat caa caa gaa att gac tcc gac cca tca gtt 259

Asp Gln Ser Ile Val Asp Gln Gln Glu Ile Asp Ser Asp Pro Ser Val

40 45 50

ctt ggc gta gta ccc caa gga tcc acc ggt atg cag gtg gtg atg ggt 307

Leu Gly Val Val Pro Gln Gly Ser Thr Gly Met Gln Val Val Met Gly

55 60 65

gga tct gtt gca aac tat tac caa gaa atc ctc aaa ctt gat gga atg 355

Gly Ser Val Ala Asn Tyr Tyr Gln Glu Ile Leu Lys Leu Asp Gly Met

70 75 80 85

aag cac ttc gcc gac ggt gaa gct aca gag agt tca tcc aag aag gaa 403

Lys His Phe Ala Asp Gly Glu Ala Thr Glu Ser Ser Ser Lys Lys Glu

90 95 100

tac ggc gga gtc cgt ggc aag tac tcg tgg att gac tac gcc ttc gag 451

Tyr Gly Gly Val Arg Gly Lys Tyr Ser Trp Ile Asp Tyr Ala Phe Glu

105 110 115

ttc ttg tct gat act ttc cga cca atc ctg tgg gcc ctg ctt ggt gcc 499

Phe Leu Ser Asp Thr Phe Arg Pro Ile Leu Trp Ala Leu Leu Gly Ala

120 125 130

tca ctg att att acc ttg ttg gtt ctt gcg gat act ttc ggt ttg caa 547

Ser Leu Ile Ile Thr Leu Leu Val Leu Ala Asp Thr Phe Gly Leu Gln

135 140 145

gac ttc cgc gct cca atg gat gag cag cct gat act tat gta ttc ctg 595

Asp Phe Arg Ala Pro Met Asp Glu Gln Pro Asp Thr Tyr Val Phe Leu

150 155 160 165

cac tcc atg tgg cgc tcg gtc ttc tac ttc ctg cca att atg gtt ggt 643

His Ser Met Trp Arg Ser Val Phe Tyr Phe Leu Pro Ile Met Val Gly

170 175 180

gcc acc gca gct cga aag ctc ggc gca aac gag tgg att ggt gca gct 691

Ala Thr Ala Ala Arg Lys Leu Gly Ala Asn Glu Trp Ile Gly Ala Ala

185 190 195

att cca gcc gca ctt ctt act cca gaa ttc ttg gca ctg ggt tct gcc 739

Ile Pro Ala Ala Leu Leu Thr Pro Glu Phe Leu Ala Leu Gly Ser Ala

200 205 210

ggc gat acc gtc aca gtc ttt ggc ctg cca atg gtt ctg aat gac tac 787

Gly Asp Thr Val Thr Val Phe Gly Leu Pro Met Val Leu Asn Asp Tyr

215 220 225

tcc gga cag gta ttc cca ccg ctg att gca gca att ggt ctg tac tgg 835

Ser Gly Gln Val Phe Pro Pro Leu Ile Ala Ala Ile Gly Leu Tyr Trp

230 235 240 245

gtg gaa aag gga ctg aag aag atc atc cct gaa gca gtc caa atg gtg 883

Val Glu Lys Gly Leu Lys Lys Ile Ile Pro Glu Ala Val Gln Met Val

250 255 260

ttc gtc cca ttc ttc tcc ctg ctg att atg atc cca gcg acc gca ttc 931

Phe Val Pro Phe Phe Ser Leu Leu Ile Met Ile Pro Ala Thr Ala Phe

265 270 275

ctg ctt gga cct ttc ggc atc ggt gtt ggt aac gga att tcc aac ctg 979

Leu Leu Gly Pro Phe Gly Ile Gly Val Gly Asn Gly Ile Ser Asn Leu

280 285 290

ctt gaa gcg att aac aac ttc agc cca ttt att ctt tcc atc gtt atc 1027

Leu Glu Ala Ile Asn Asn Phe Ser Pro Phe Ile Leu Ser Ile Val Ile

295 300 305

cca ttg ctc tac cca ttc ttg gtt cca ctt gga ttg cac tgg cca cta 1075

Pro Leu Leu Tyr Pro Phe Leu Val Pro Leu Gly Leu His Trp Pro Leu

310 315 320 325

aac gcc atc atg atc cag aac atc aac acc ctg ggt tac gac ttc att 1123

Asn Ala Ile Met Ile Gln Asn Ile Asn Thr Leu Gly Tyr Asp Phe Ile

330 335 340

cag gga cca atg ggt gcc tgg aac ttc gcc tgc ttc ggc ctg gtc acc 1171

Gln Gly Pro Met Gly Ala Trp Asn Phe Ala Cys Phe Gly Leu Val Thr

345 350 355

ggc gtg ttc ttg ctc tcc att aag gaa cga aac aag gcc atg cgt cag 1219

Gly Val Phe Leu Leu Ser Ile Lys Glu Arg Asn Lys Ala Met Arg Gln

360 365 370

gtt tcc ctg ggt ggc atg ttg gct ggt ttg ctc ggc ggc att tcc gag 1267

Val Ser Leu Gly Gly Met Leu Ala Gly Leu Leu Gly Gly Ile Ser Glu

375 380 385

cct tcc ctc tac ggt gtt ctg ctc cga ttc aag aag acc tac ttc cgc 1315

Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys Lys Thr Tyr Phe Arg

390 395 400 405

ctc ctg ccg ggt tgt ttg gca gca 1339

Leu Leu Pro Gly Cys Leu Ala Ala

410

<210>32

<211>413

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>32

Met Ala Ser Lys Leu Thr Thr Thr Ser Gln His Ile Leu Glu Asn Leu

1 5 10 15

Gly Gly Pro Asp Asn Ile Thr Ser Met Thr His Cys Ala Thr Arg Leu

20 25 30

Arg Phe Gln Val Lys Asp Gln Ser Ile Val Asp Gln Gln Glu Ile Asp

35 40 45

Ser Asp Pro Ser Val Leu Gly Val Val Pro Gln Gly Ser Thr Gly Met

50 55 60

Gln Val Val Met Gly Gly Ser Val Ala Asn Tyr Tyr Gln Glu Ile Leu

65 70 75 80

Lys Leu Asp Gly Met Lys His Phe Ala Asp Gly Glu Ala Thr Glu Ser

85 90 95

Ser Ser Lys Lys Glu Tyr Gly Gly Val Arg Gly Lys Tyr Ser Trp Ile

100 105 110

Asp Tyr Ala Phe Glu Phe Leu Ser Asp Thr Phe Arg Pro Ile Leu Trp

115 120 125

Ala Leu Leu Gly Ala Ser Leu Ile Ile Thr Leu Leu Val Leu Ala Asp

130 135 140

Thr Phe Gly Leu Gln Asp Phe Arg Ala Pro Met Asp Glu Gln Pro Asp

145 150 155 160

Thr Tyr Val Phe Leu His Ser Met Trp Arg Ser Val Phe Tyr Phe Leu

165 170 175

Pro Ile Met Val Gly Ala Thr Ala Ala Arg Lys Leu Gly Ala Asn Glu

180 185 190

Trp Ile Gly Ala Ala Ile Pro Ala Ala Leu Leu Thr Pro Glu Phe Leu

195 200 205

Ala Leu Gly Ser Ala Gly Asp Thr Val Thr Val Phe Gly Leu Pro Met

210 215 220

Val Leu Asn Asp Tyr Ser Gly Gln Val Phe Pro Pro Leu Ile Ala Ala

225 230 235 240

Ile Gly Leu Tyr Trp Val Glu Lys Gly Leu Lys Lys Ile Ile Pro Glu

245 250 255

Ala Val Gln Met Val Phe Val Pro Phe Phe Ser Leu Leu Ile Met Ile

260 265 270

Pro Ala Thr Ala Phe Leu Leu Gly Pro Phe Gly Ile Gly Val Gly Asn

275 280 285

Gly Ile Ser Asn Leu Leu Glu Ala Ile Asn Asn Phe Ser Pro Phe Ile

290 295 300

Leu Ser Ile Val Ile Pro Leu Leu Tyr Pro Phe Leu Val Pro Leu Gly

305 310 315 320

Leu His Trp Pro Leu Asn Ala Ile Met Ile Gln Asn Ile Asn Thr Leu

325 330 335

Gly Tyr Asp Phe Ile Gln Gly Pro Met Gly Ala Trp Asn Phe Ala Cys

340 345 350

Phe Gly Leu Val Thr Gly Val Phe Leu Leu Ser Ile Lys Glu Arg Asn

355 360 365

Lys Ala Met Arg Gln Val Ser Leu Gly Gly Met Leu Ala Gly Leu Leu

370 375 380

Gly Gly Ile Ser Glu Pro Ser Leu Tyr Gly Val Leu Leu Arg Phe Lys

385 390 395 400

Lys Thr Tyr Phe Arg Leu Leu Pro Gly Cys Leu Ala Ala

405 410

<210>33

<211>428

<212>DNA

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(1)..(405)

<223>FRXA01943

<400>33

cct gac cca atc ttt gca gca ggc aag ctt gga cca ggc att gca atc 48

Pro Asp Pro Ile Phe Ala Ala Gly Lys Leu Gly Pro Gly Ile Ala Ile

1 5 10 15

caa cca act gga aac acc gtt gtt gct cca gca gac gct act gtc atc 96

Gln Pro Thr Gly Asn Thr Val Val Ala Pro Ala Asp Ala Thr Val Ile

20 25 30

ctt gtc cag aaa tct gga cac gca gtg gca ttg cgc tta gat agc gga 144

Leu Val Gln Lys Ser Gly His Ala Val Ala Leu Arg Leu Asp Ser Gly

35 40 45

gtt gaa atc ctt gtc cac gtt gga ttg gac acc gtg caa ttg ggc ggc 192

Val Glu Ile Leu Val His Val Gly Leu Asp Thr Val Gln Leu Gly Gly

50 55 60

gaa ggc ttc acc gtt cac gtt gag cgc agg cag caa gtc aag gcg ggg 240

Glu Gly Phe Thr Val His Val Glu Arg Arg Gln Gln Val Lys Ala Gly

65 70 75 80

gat cca ctg atc act ttt gac gct gac ttc att cga tcc aag gat cta 288

Asp Pro Leu Ile Thr Phe Asp Ala Asp Phe Ile Arg Ser Lys Asp Leu

85 90 95

cct ttg atc acc cca gtt gtg gtg tct aac gcc gcg aaa ttc ggt gaa 336

Pro Leu Ile Thr Pro Val Val Val Ser Asn Ala Ala Lys Phe Gly Glu

100 105 110

att gaa ggt att cct gca gat cag gca aat tct tcc acg act gtg atc 384

Ile Glu Gly Ile Pro Ala Asp Gln Ala Asn Ser Ser Thr Thr Val Ile

115 120 125

aag gtc aac ggc aag aac gag taacctggga tccatgttgc gca 428

Lys Val Asn Gly Lys Asa Glu

130 135

<210>34

<211>135

<212>PRT

<213>Corynebacterium glutamicum (Corynebacterium glutamicum)

<400>34

Pro Asp Pro Ile Phe Ala Ala Gly Lys Leu Gly Pro Gly Ile Ala Ile

1 5 10 15

Gln Pro Thr Gly Asn Thr Val Val Ala Pro Ala Asp Ala Thr Val Ile

20 25 30

Leu Val Gln Lys Ser Gly His Ala Val Ala Leu Arg Leu Asp Ser Gly

35 40 45

Val Glu Ile Leu Val His Val Gly Leu Asp Thr Val Gln Leu Gly Gly

50 55 60

Glu Gly Phe Thr Val His Val Glu Arg Arg Gln Gln Val Lys Ala Gly

65 70 75 80

Asp Pro Leu Ile Thr Phe Asp Ala Asp Phe Ile Arg Ser Lys Asp Leu

85 90 95

Pro Leu Ile Thr Pro Val Val Val Ser Asn Ala Ala Lys Phe Gly Glu

100 105 110

Ile Glu Gly Ile Pro Ala Asp Gln Ala Asn Ser Ser Thr Thr Val Ile

115 120 125

Lys Val Asn Gly Lys Asn Glu

130 135

<210>35

<211>18

<212>DNA

<213>artificial sequence

<220>

<223>artificial sequence note: primer

<400>35

ggaaacagta tgaccatg 18

<210>36

<211>17

<212>DNA

<213>artificial sequence

<220>

<223>artificial sequence note: primer

<400>36

gtaaaacgac ggccagt 17

Claims

1. the isolated nucleic acid molecules or its complementary series of the nucleotide sequence comprising SEQ ID NO:21.

2. the isolated nucleic acid molecules or its complementary series of the polypeptide of amino acid sequence of the coding comprising SEQ ID NO:22.

3. the isolated nucleic acid molecules or its complementary series of the naturally occurring allelic variant of the polypeptide of amino acid sequence of the coding comprising SEQ ID NO:22.

4. isolated nucleic acid molecules, comprising having at least nucleotide sequence of 50% identity or its complementary series with the whole nucleotide sequence of SEQ ID NO:21.

5. isolated nucleic acid molecules, it includes the segments or its complementary series of at least 15 continuous nucleotides of the nucleotide sequence of SEQ ID NO:21.

6. isolated nucleic acid molecules, coding has at least polypeptide of the amino acid sequence of 50% identity or its complementary series comprising the complete amino acid sequence with SEQ ID NO:22.

7. isolated nucleic acid molecules, the nucleotide sequence of the nucleic acid molecules comprising any one of claim 1-6 and encoding heterologous polypeptide.

8. the carrier of the nucleic acid molecules comprising any one of claim 1-7.

9. the carrier of claim 8, which is expression vector.

10. the host cell that the expression vector of claim 9 transfects.

11. the host cell of claim 10, wherein the cell is microorganism.

12. the host cell of claim 11, wherein the cell belongs to Corynebacterium or brevibacterium.

13. the host cell of claim 10, wherein the expression of described nucleic acid molecules, the adjusting for causing the cell fine chemical to produce.

14. the host cell of claim 14, wherein described fine chemical is selected from following substance: organic acid, proteinogenic amino acids, nonprotein source amino acid, purine bases and pyrimidine bases, nucleosides, nucleotide, lipid, saturation and unsaturated fatty acid, glycol, carbohydrate, aromatic compound, vitamin, co-factor, polyketide and enzyme.

15. the method for producing polypeptide, including cultivating the host cell of claim 10 in suitable culture medium, to produce polypeptide.

16. isolated polypeptide, the amino acid sequence comprising SEQ ID NO:22.

17. isolated polypeptide, the naturally occurring allelic variant of the polypeptide comprising the amino acid sequence containing SEQ ID NO:22.

18. isolated polypeptide, by a kind of nucleic acid molecule encoding, which includes the nucleotide sequence for having at least 50% identity with the whole nucleotide sequence of SEQ ID NO:21.

19. isolated polypeptide, comprising there is the amino acid sequence of at least 50% identity with the complete amino acid sequence of SEQ ID NO:22.

20. isolated polypeptide, the segment of the polypeptide comprising the amino acid sequence containing SEQ ID NO:22, wherein the polypeptide fragment keeps the biological activity of the polypeptide of the amino acid sequence containing SEQ ID NO:22.

21. isolated polypeptide, the amino acid sequence comprising the nucleic acid molecule encoding by the nucleotide sequence comprising SEQ ID NO:21.

22. the isolated polypeptide of any one of claim 16-21, further includes heterologous amino acid sequence.

23. the method for producing fine chemical, the cell including cultivating claim 10, to generate fine chemical.

24. the method for claim 23, wherein described method also comprises the step of recycling fine chemical from described culture.

25. the method for claim 23, wherein described cell belongs to Corynebacterium or brevibacterium.

26. the method for claim 23, wherein described cell is selected from the group: Corynebacterium glutamicum, Corynebacterium herculis, lily bar bacterium, Corynebacterium acctoacidophlum, vinegar paddy bar bacterium, thermophilic acetyl bar bacterium, corynebacterium ammoniagenes, Corynebacterium fujiokense, Corynebacteriumnitrilophilus, brevibacterium ammoniagene, Brevibacterium butanicum, disagreement brevibacterium, brevibacterium flavum, Xi Shi brevibacterium, ketoglutaric acid brevibacterium, Brevibacteriumketosoreductum, brevibacterium, extension brevibacterium, solve bacterial strain listed by paraffin brevibacterium and table 3.

27. the method for claim 23, wherein the expression of the nucleic acid molecules of described carrier, the adjusting for causing the cell fine chemical to produce.

28. the method for claim 23, wherein described fine chemical is selected from following substance: organic acid, proteinogenic amino acids, nonprotein source amino acid, purine bases and pyrimidine bases, nucleosides, nucleotide, lipid, saturation and unsaturated fatty acid, glycol, carbohydrate, aromatic compound, vitamin, co-factor, polyketide and enzyme.

29. the method for claim 23, wherein described fine chemical is amino acid.

30. the method for claim 29, wherein described amino acid is selected from following amino acid: lysine, glutamic acid, glutamine, alanine, aspartic acid, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine, tryptophan.

31. the method for producing fine chemical, including such cell is cultivated, the genomic DNA of the cell has been inserted into the nucleic acid molecules of any one of claim 1-6 and has changed.

32. diagnosing the presence or active method of corynebacterium diphtheriae in subject, the presence of the peptide molecule of at least one nucleic acid molecules or claim 16-21 including claim 1-6 in detection subject, to diagnose the presence or activity of corynebacterium diphtheriae in subject.

33. the host cell of the nucleic acid molecules containing SEQ ID NO:21, wherein the nucleic acid molecules are destroyed.

34. the host cell of the nucleic acid molecules containing SEQ ID NO:21, nucleic acid molecules therein, which contain one or more, modifies the nucleic acid for listing sequence in SEQ ID NO:21.

35. the host cell of the nucleic acid molecules containing SEQ ID NO:21, wherein the nucleic acid molecules adjustment region is modified relative to the molecule wild type adjustment region.