CN101130778A

CN101130778A - Corynebacterium glutamicum gene encoding hpr of phosphoenolpyruvate:sugar phosphotransferase system

Info

Publication number: CN101130778A
Application number: CNA2007100920738A
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: 2008-02-27
Also published as: CN101054593A; ZA200200816B; CN101054592A

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

Coding phosphoenolpyruvic acid: the proteinic Corynebacterium glutamicum gene of carbohydrate phosphotransferase system

The application is to be the dividing an application of Chinese patent application 00812165.6 " coding phosphoenolpyruvic acid: the proteinic Corynebacterium glutamicum gene of carbohydrate phosphotransferase system " on June 27th, 2000 applying date.

Technical field

The present invention relates to the nucleic acid molecule of the new Corynebacterium glutamicum pts protein of separated coding.The present invention also relates to antisense nucleic acid molecule, contain the recombinant expression vector of PTS nucleic acid molecule, and the host cell that has imported expression vector.The present invention also further relates to isolating pts protein, PTS mutain, fused protein, antigen peptide, and the method that improves the required compound production of being undertaken by this organism based on Corynebacterium glutamicum PTS gene genetic engineering.

Background technology

Specific product in the cell in the naturally occurring metabolic process and by product have purposes in a lot of industries, comprise food, feed, makeup and pharmaceutical industries.These molecules are generically and collectively referred to as " fine chemistry material ", comprise organic acid, protein source and amino acid non-protein source, Nucleotide and nucleosides, lipid and lipid acid, glycol, carbohydrate, aromatics, VITAMIN and cofactor and enzyme.Can produce the bacterium of a large amount of specific desired molecules of justacrine by large scale culturing, these products of the preparation of most convenient.A kind of useful especially organism that is used for this purpose is exactly Corynebacterium glutamicum (Corynebacterium glutamicum), a kind of Gram-positive nonpathogenic bacteria.By bacterial strain screening, the mutant strain of the large quantities of required compounds of many generations has appearred.Yet,, be a consuming time and difficult process for improving the bacterial strain screening that particular molecule production is carried out.

Summary of the invention

The invention provides new bacterial nucleic acid molecule, these molecules serve many purposes.These purposes comprise evaluation can produce the microorganism of fine chemistry material, regulate the somatotype of generation, Corynebacterium glutamicum or relationship bacterium of the fine chemistry material in Corynebacterium glutamicum or the relationship bacterium and evaluation, as the reference point of drawing Corynebacterium glutamicum gene picture group spectrum.The nucleic acid molecule encoding protein that these are new is called phosphoenolpyruvic acid herein: carbohydrate phosphotransferase system (PTS) protein.

Corynebacterium glutamicum is a kind of Gram-positive aerobic bacteria, is used to the various fine chemistry materials of scale operation usually in industry, also is used for degradation of hydrocarbon (for example in Oil spills) and oxidation terpinol.Therefore, PTS nucleic acid molecule of the present invention can be used to identify the microorganism that can be used to produce the fine chemistry material, for example passes through fermentation process.Regulate the expression of PTS nucleic acid molecule of the present invention, perhaps modify the sequence of PTS nucleic acid molecule of the present invention, can be used for regulating the production (for example, improving the generation of one or more fine chemistry materials in excellent bacillus or the tyrothricin) of one or more fine chemistry materials of microorganism.

PTS nucleic acid molecule of the present invention can be used for identifying whether a kind of microorganism is Corynebacterium glutamicum or its relationship bacterial strain, the perhaps existence of Corynebacterium glutamicum or its relationship bacterial strain in the Identifying micro-organisms population mixture.The invention provides the nucleotide sequence of many Corynebacterium glutamicum genes; Under stringent condition, detect the genomic dna that from single microorganism or mixing microorganisms colony culture, extracts with probe, this probe has covered the distinctive one section zone of Corynebacterium glutamicum gene, can determine whether that this microorganism exists.Although the right and wrong of Corynebacterium glutamicum own are pathogenic, it is relevant with the pathogen species in the human body, for example diphtheria corynebacterium (Corynebacterium diphtheriae) (diphtheria is caused a disease former); Survey this microorganism and have great clinical practice.

PTS nucleic acid molecule of the present invention also can perhaps be drawn the reference point of its relationship strain gene picture group spectrum as the reference point of drawing Corynebacterium glutamicum gene picture group spectrum.Similar, these molecules, perhaps its variant or its part can be as the genetic markers of genetic engineering rod bacillus or tyrothricin.

For example, the pts protein of new nucleic acid molecule encoding of the present invention can perhaps participate in signal conduction in the cell in this microorganism similarly being that the such high-energy carbon-containing molecules of glucose is transported into Corynebacterium glutamicum.Consider the practicality of the cloning vector that can in Corynebacterium glutamicum, use, for example at Sinskey et al., United States Patent (USP) numbering No.4,649, disclosed in 119, and consider Genetic Manipulative Technology (Yashihama et al, the J.Bacteriol.162:591-597 (1985) of Corynebacterium glutamicum and relationship tyrothricin bacterial classification (for example brevibacterium); Katsumata et al., J.Bacteriol.159:306-311 (1984); And Santamaria et al., J.Gen.Microbiol.130:2237-2246 (1984)), nucleic acid molecule of the present invention can be used for the genetic engineering of this organism, makes it to become the better or more effective producer of one or more fine chemistry materials.

Can modify PTS molecule of the present invention, thereby make output, production and/or the production efficiency of one or more fine chemistry materials be improved.For example,, can optimize its activity, make and are transported glucose uptake quantity or glucose into the speed of cell is improved by modifying a kind of pts protein that participates in glucose uptake.The degraded of glucose or other carbohydrates can provide energy in the cell, and these energy can be used for promoting the disadvantageous biochemical reaction of energy, and for example those relate to the biosynthetic reaction of fine chemistry material.Degraded also provides some necessary intermediate of fine chemistry material biosynthesizing or precursor molecule, for example amino acid, VITAMIN and cofactor.By modifying PTS molecule of the present invention to increase the quantity of high-energy carbon molecule in the cell, both can increase and finish the energy supply of producing the necessary pathways metabolism of one or more fine chemistry materials, can increase the needed endocellular metabolism material of this production storehouse again.

In addition, PTS molecule of the present invention can participate in signal transduction path in the one or more of cells, and these signal transduction paths can influence the output and/or the throughput rate of one or more fine chemistry materials in the Corynebacterium glutamicum.For example, in case there is the carbohydrate of sufficient amount in the cell, the necessary protein of one or more carbohydrates of input (for example from extracellular medium, HPr, Enzyme I, the perhaps a kind of composition in the Enzyme II complex body) often is translated the back and modifies, thereby they can not be input to sugar in the cell again.The quantity of sugar when movement system is closed may be enough for keeping the cell normal function, and this may limit the excessive production of required chemical substance.Therefore, it is very worthwhile modifying pts protein of the present invention and making it no longer include reaction to this negative adjusting, so, allow interior one or more sugar of cell to reach higher concentration, and extend by reaction, from the organism that contains this pts protein mutant, just can more effectively produce or one or more fine chemistry materials of more large-tonnage acquisition.

The invention provides the nucleic acid molecule of new coded protein, this protein is called phosphoenolpyruvic acid herein: carbohydrate phosphotransferase system (PTS) protein, they the high-energy carbon molecule (for example participate in, glucose, fructose, sucrose) the input Corynebacterium glutamicum, and/or participate in signal transduction path in the one or more of Corynebacterium glutamicum cells.These proteinic examples comprise the protein of the coded by said gene that those are listed in table 1.

Therefore, one aspect of the present invention relates to, the nucleic acid molecule that separates the nucleotide sequence contain one section a kind of pts protein of coding or its biologically-active moiety (for example, cDNA, DNA, and separate to be suitable as and survey or the primer of amplification PTS coding nucleic acid (for example DNA or RNA) or the nucleic acid fragment of hybridization probe perhaps RNA).In particularly preferred embodiments, the nucleotide sequence that it is odd number that isolated nucleic acid molecule comprises one section sequence number that is listed in the sequence table (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) or coding region or its complementary sequence of a this nucleotide sequence.In other particularly preferred embodiments, isolating nucleic acid molecule of the present invention comprise with sequence table in sequence number be odd number nucleotide sequence (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) or its part about at least 50% homology is arranged, at least about 60% homology is preferably arranged, and preferred have about at least 70%, 80%, or 90% homology, even preferredly have about at least 95%, 96%, 97%, 98%, 99% or higher homology.In other embodiment preferred, the isolated nucleic acid molecule coding is listed in the even number sequence number aminoacid sequence (for example, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8) in the sequence table.The preferred pts protein of the present invention also preferably has at least a PTS activity described herein.

In another embodiment, encode a kind of protein or its part of isolated nucleic acid molecule, protein wherein or its part comprise one section aminoacid sequence, this sequence and aminoacid sequence of the present invention are (for example, the sequence of even number sequence number in sequence table) sufficient homology is arranged, for example, sufficient homology is arranged and make this protein or its part have the PTS activity with aminoacid sequence of the present invention.Preferably, the protein of nucleic acid molecule encoding or its part, have the ability that participates in high-energy carbon molecule (for example, glucose, fructose, sucrose) input Corynebacterium glutamicum, and/or participate in the ability of signal transduction path in the one or more of Corynebacterium glutamicum cells.In one embodiment, a kind of protein of nucleic acid molecule encoding and aminoacid sequence of the present invention (for example, the complete amino acid sequence of selecting in the even number sequence number sequence from sequence table) have about at least 50% homology, and about at least 60% homology is preferably arranged, preferred have about at least 70%, 80%, 90% homology, most preferred have about at least 95%, 96%, 97%, 98%, 99% or higher homology.In another preferred embodiment, protein is the Corynebacterium glutamicum protein of total length, this protein and full length amino acid sequence of the present invention (by be presented in the corresponding sequence table the odd serial numbers nucleotide sequence (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7) the open reading frame coding) abundant homology.

In another preferred embodiment, isolated nucleic acid molecule is from Corynebacterium glutamicum, and a kind of protein of encoding (for example a kind of PTS fusion rotein), this protein comprises one section biological activity zone, this zone and a kind of aminoacid sequence of the present invention are (for example, a sequence in the sequence table even number sequence number sequence) has about at least 50% or higher homology, and this protein (for example can participate in a high-energy carbon molecule, glucose, fructose, sucrose) input Corynebacterium glutamicum, and/or participate in signal transduction path in the one or more of Corynebacterium glutamicum cells, perhaps have one or more and be listed in activity in the table 1, and this protein also includes the heterologous nucleic acid sequence of one section coding heterologous polypeptide or regulation domain.

In another embodiment, isolated nucleic acid molecule has the length of 15 Nucleotide at least, and under stringent condition with contain the making nucleic acid molecular hybridization of nucleotide sequence of the present invention (for example, odd serial numbers sequence in sequence table).Preferably, isolated nucleic acid molecule is consistent with naturally occurring nucleic acid molecule.Be more preferably isolated nucleic acid molecule encode naturally occurring Corynebacterium glutamicum pts protein, perhaps its biologically-active moiety.

Another aspect of the present invention relates to carrier, for example contains the recombinant expression vector of nucleic acid molecule of the present invention and is introduced into the host cell of this carrier.In one embodiment, by cultivating in suitable medium, this host cell is used to produce pts protein.Can from substratum or host cell, separate this pts protein then.

In addition, another aspect of the present invention relates to a kind of microorganism through hereditary change, and the PTS gene has been introduced into wherein or and has been changed.In one embodiment, by introducing nucleic acid molecule of the present invention, changed the genome of this microorganism as genetically modified encoding wild type or mutant PTS sequence.In another embodiment, changed the endogenous PTS gene in this microbial genome, for example, carried out functional destruction by using the PTS gene that has changed to carry out homologous recombination.In another embodiment, PTS gene endogenous or that introduce is changed by one or more point mutation, disappearance or inversion in this microorganism, but still can the encoding function pts protein.In another embodiment, change one or more regulation domain (for example, promotor, repressor or inductor) of microorganism PTS gene, thereby regulate the PTS expression of gene.In preferred embodiments, microorganism belongs to excellent bacillus specie or quarter butt bacterial classification, especially preferably Corynebacterium glutamicum.In preferred embodiments, also use the required compound of microorganisms producing, for example amino acid, especially preferably Methionin.

On the other hand, the invention provides corynebacterium diphtheriae existence or active method among a kind of evaluation experimenter.This method comprises the detection to one or more nucleic acid of the present invention or aminoacid sequence among the experimenter (for example, being listed in the sequence of SEQ ID NO 1 to 34 in the sequence table), thereby can detect existence or the activity of Corynebacterium glutamicum among the experimenter.

In addition, another aspect of the present invention relates to and isolates pts protein or its part, for example its biologically-active moiety.In a preferred embodiment, isolating pts protein or its part (for example can participate in a high-energy carbon molecule, glucose, fructose, sucrose) the input Corynebacterium glutamicum, and also can participate in signal transduction path in the one or more of Corynebacterium glutamicum cells.In another preferred embodiment, isolating its part of pts protein person and a kind of aminoacid sequence of the present invention are (for example, sequence table even number sequence number a: sequence in the sequence) sufficiently high homology is arranged, make this protein or its part (for example can participate in a high-energy carbon molecule, glucose, fructose, sucrose) import the ability of Corynebacterium glutamicum, and/or also participate in the ability of signal transduction path in the one or more of Corynebacterium glutamicum cells.

The present invention also provides the Separation Product of pts protein.In preferred embodiments, pts protein comprises aminoacid sequence of the present invention (for example, sequence table even number sequence number a: sequence in the sequence).In another preferred embodiment, the present invention is relevant with isolating full length protein, and (sequence table even number sequence number: a sequence in the sequence) (is the open reading frame coding of odd number by the sequence number that is presented in the corresponding sequence table) has quite high homology for this protein and complete aminoacid sequence of the present invention.In addition, in another embodiment, protein and complete aminoacid sequence of the present invention are (for example, even number sequence number sequence in the sequence table) about at least 50% homology is arranged, at least about 60% homology is preferably arranged, and preferred have about at least 70%, 80%, or 90% homology, most preferred have about at least 95%, 96%, 97%, 98%, or 99% or higher homology.In another embodiment, an aminoacid sequence that isolating pts protein comprises and an aminoacid sequence of the present invention are (for example, an even number sequence number sequence in the sequence table) has about at least 50% or higher homology, and (for example can participate in a high-energy carbon molecule, glucose, fructose, sucrose) the input Corynebacterium glutamicum, with/and participate in signal transduction path in the one or more of Corynebacterium glutamicum cells, perhaps have one or more activity of listing in the table 1.

In addition, isolating pts protein can contain the aminoacid sequence by nucleic acid sequence encoding, this nucleotide sequence and a nucleic acid array hybridizing that is listed in the even number sequence number in the sequence table, hybridize under stringent condition for example, at least about 50% homology is perhaps arranged with this nucleotide sequence, at least about 60% homology is preferably arranged, preferred have about at least 70%, 80%, or 90% homology, even preferredly have about at least 95%, 96%, 97%, 98%, or 99% or higher homology.The preferred form of pts protein has one or more biological activitys described herein equally, also is preferred.

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

On the other hand, the invention provides the method that screening can be regulated the active molecule of pts protein.This molecule by with protein molecule itself or substrate interaction, perhaps combine, perhaps by regulating transcribing or translate and regulating the pts protein activity of PTS nucleic acid molecule of the present invention with the mating partner of pts protein.

Another aspect of the present invention relates to the method for producing the fine chemistry material.This method relates to cultivates the cell that contains a kind of carrier, and this carrier instructs the expression of PTS nucleic acid molecule of the present invention, thereby produces the fine chemistry material.In a preferred embodiment, this method also comprises the step that obtains to contain this carrier cell, in this step, and the carrier transfectional cell that use can instruct the PTS nucleic acid molecule to express.In another preferred embodiment, this method also comprises the step that reclaims the fine chemistry material from substratum.In an especially preferred embodiment, cell is excellent bacillus specie or quarter butt bacterial classification, perhaps is selected from those bacterial strains that are listed in the table 3.

Another aspect of the present invention relates to the method that molecule produces in the microorganism of regulating.This method comprises medicament exposing cell that use to regulate pts protein activity or PTS expression of nucleic acid, makes the activity of related activity when lacking this medicament of cell that change take place.In a preferred embodiment,, regulate cell, make the output of the required fine chemistry material of this microorganism or generation efficient be improved in order to absorb one or more carbohydrates.Regulating the active medicament of pts protein, can be the medicament that stimulates pts protein activity or PTS expression of nucleic acid.Stimulate the example of the medicament of pts protein activity or PTS expression of nucleic acid, comprise small molecules, active pts protein and the nucleic acid of the pts protein of transfered cell of encoding.The example of the medicament that suppresses the pts protein activity or express comprises small molecules and antisense PTS nucleic acid molecule.

Another aspect of the present invention relates to the method for regulating required compound output in the cell, comprises that wild-type or mutant PTS gene transfered cell this gene or be retained on the independent plasmid perhaps is incorporated in the host cell gene group.If be incorporated in the host cell gene group, this integration can be arbitrarily, or takes place by homologous recombination, thereby makes the copy of introducing to replace natural gene, causes in the cell generation of required compound adjusted.In a preferred embodiment, this output obtains increasing.In another preferred embodiment, said fine chemistry material is an amino acid.In an especially preferred embodiment, said amino acid is L-Methionin.

Embodiment

The invention provides PTS nucleic acid and protein molecule, they participate in Corynebacterium glutamicum picked-up high-energy carbon molecule (for example, glucose, fructose, sucrose), and also can participate in the interior signal transduction path of one or more of cells in this microorganism.Molecule of the present invention can be used for regulating the production of fine chemistry material in the microorganism.This adjusting may be the increase owing to the high energy molecular level of required generation in the cell, for example, ATP, it similarly is the molecule of the so unfavorable biochemical reaction of energy of fine chemistry material biosynthesizing that GTP and other are used to promote.The adjusting of fine chemistry material production also may be owing to this fact, and promptly the degraded product of a lot of carbohydrates is used as the intermediate or the precursor of other biological route of synthesis, comprises the biosynthetic pathway of some fine chemistry material.In addition, known pts protein participates in signal transduction path in some cell, and they may have the activity of adjusting to the pathways metabolism of one or more of fine chemistry materials; By utilizing these pts proteins, can activate the biosynthetic pathway of fine chemistry material or suppress its chemical degradation approach.All respects of the present invention further describe as follows.

I. fine chemistry material

" fine chemistry material " this speech is well known in the art, comprises the molecule that uses in various industries that organism produces, for example but not only be confined to pharmacy, agricultural and makeup industry.This compound comprises organic acid, tartrate for example, methylene-succinic acid and diaminopimelic acid, protein source and nonprotein source amino acid, purine bases and pyrimidine bases, nucleosides, and Nucleotide (for example similarly is to be described in Kuninaka, A. (1996) Nucleotides and related compounds, p.561-612, in Biotechnology vol.6, Rehm et al., in eds.VCH:Weinheim and the contained reference thereof), lipid, saturated and unsaturated fatty acids (for example arachidonic acid), glycol (for example, propane diol and butanediol), aromatics (for example, aromatic amine, Vanillin and indigo), VITAMIN and cofactor are (referring to Ullmann ' s Encyclopedia of IndustrialChemistry, vol.A27, " Vitamins ", p.443-613 (1996) VCH:Weinheim and referencestherein; And Ong, A.S., Niki, E.﹠amp; 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, AOCS Press, (1995)), enzyme, polyketide (ployketides) (Cane et al., (1998) Science 282:63-68), and all are at Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, the chemical substance of describing in ISBN:0818805086 and the reference thereof.The metabolism of some these fine chemistry material and purposes further describe as follows.

A. amino acid whose metabolism and purposes

Amino acid comprises the basic structural unit of all proteins, equally also is that all organism normal cell biological functions are necessary." amino acid " this speech is well known in the art.The amino acid of protein source has 20 kinds, it is proteinic structural unit, be connected by peptide bond each other, but not protein source amino acid (known have hundreds of kind) generally can not appear in the protein (referring to Ulmann ' s Encyclopedia of Industrial Chemistry, vol.A2, p.57-97 VCH:Weinheim (1985)).Though the normally natural unique type that exists in the protein of L-amino acid, amino acid can be D-or L-optical configuration.Each biosynthesizing or degradation pathway in 20 kinds of protein source amino acid, all in prokaryotic cell prokaryocyte or eukaryotic cell, have separately characteristics (for example referring to Stryer, L.Biochemistry, 3 ^RdEdition, pages 578-590 (1988)).Why like this " essential " amino acid (Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, Threonine, tryptophane and Xie Ansuan) name, be that they can be converted into remaining 11 kinds of " nonessential " amino acid (L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, L-glutamic acid, glutamine, glycine, proline(Pro), Serine, tyrosine) by simple biosynthetic pathway because these amino acid bios synthesize complicated normally essential nutritional condition.Though higher organism has more synthetic this amino acid whose abilities really, for normal protein synthesis must replenish indispensable amino acid from diet.

They are except the function in protein synthesis, and these amino acid just himself are interesting chemical substances, and much have various application in them in food, feed, chemistry, makeup, agricultural and pharmaceutical industries.Methionin is a kind of important amino acid for the mankind not only aspect nutrition, and for similarly being that the such monogastric animal of poultry and pig also is important.L-glutamic acid is that (msg powder type MSG), and is widely used in whole food industries, as aspartic acid, glycine, halfcystine to the most frequently used flavor additives useful.Glycine, L-methionine(Met) and tryptophane all are used for pharmaceutical industries.Glutamine, Xie Ansuan, leucine, Isoleucine, Histidine, arginine, proline(Pro), Serine and L-Ala all are applied in pharmaceutical industries and the makeup industry.Threonine, tryptophane and D/L-methionine(Met) are the fodder additives (Leuchtenberger that uses always, 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 also are of great use as the precursor of synthesizing amino acid and protein synthesis, N-acetylcystein for example, S-carboxymethyl-L-halfcystine, (S)-5-hydroxyryptophan, and other are at Ulmann ' s Encyclopedia of Industrial Chemistry, vol.A2, p.57-97 VCH:Weinheim, the molecule of describing in 1985.

In the organism that can produce natural amino acid, bacterium for example, these natural amino acid whose biosynthesizing solve very abundant (amino acid whose biosynthesizing of bacterium and adjusting thereof, referring to Umbarger, H.E. (1978) Ann.Rev.Biochem.47:533-606).Aspartic acid is synthetic by the ammonification of α-Tong Wuersuan reduced form, and the latter is the intermediate of tricarboxylic acid cycle.Glutamine, proline(Pro) and arginine are all produced successively by L-glutamic acid.The biosynthesizing of Serine is the process in one three step, starts from 3-phoshoglyceric acid (glucolytic intermediate), after peroxidation, transamination, each step of hydrolytic action, ends at this amino acid.Halfcystine and glycine are all produced by Serine; The former is formed by homocysteine and Serine condensation, and the latter transfers to tetrahydrofolic acid (THFA) to the side chain beta carbon and obtains, and this reaction is catalytic by serine hydroxymethylase.Phenylalanine and tyrosine, synthetic in the biosynthetic pathway in one 9 step by erythrose-4-phosphate and phosphoenolpyruvic acid, they are precursors of glycolytic pathway and pentose-phosphate pathway, these two approach are just different after synthetic prephenic acid.Tryptophane also can be produced by these two kinds initial molecules, but its synthetic be the approach in one 11 step.Tyrosine also can be synthetic by phenylalanine, and its reaction is catalytic by Phenylalanine hydroxylase.L-Ala, Xie Ansuan and leucine all are the biosynthetic products of glycolysis-end product pyruvic acid.Aspartic acid is synthetic by oxaloacetic acid, and the latter is the intermediate of tricarboxylic acid cycle.L-asparagine, methionine(Met), Threonine and Methionin all are transformed by aspartic acid.Isoleucine is formed by Threonine.By 9 approach that go on foot of a complexity, can be from a kind of active sugar, ribose 5-phosphate-1-tetra-sodium produces Histidine.

Exceeding the synthetic required amino acid of cell protein can not store, but (comment is referring to Stryer, and L.Biochemistry 3 for the cell main metabolic pathway provides intermediate after being degraded ^RdEd.Ch.21 " Amino Acid Degradation and the Urea Cycle " p.495-516 (1988)).Although it is useful metabolism intermediate that cell can transform unnecessary amino acid, produces amino acid and will consume a lot of energy, precursor molecule and synthetic required enzyme.Therefore it is not astonishing regulating amino acid whose biosynthesizing with feedback inhibition, the existence of special acid can slow down or the generation that stops himself fully (for the comment of amino acid biosynthetic pathway Feedback mechanism, referring to Stryer, L.Biochemistry 3 ^RdEd.Ch.24 " Biosynthesis of Amino Acid and Heme " p.575-600 (1988)).Therefore, the amino acid quantity that all existed in the cell of the output of any specific amino acids limits.

B. the metabolism of VITAMIN, cofactor and dietetic product and purposes

VITAMIN, cofactor and dietetic product comprise another component, though other biological, for example bacterium can be synthesized these molecules, higher animal lost synthetic they ability and can only absorb.These molecules or itself are biologically active substances, or the precursor of biologically active substance, and this biologically active substance can be the intermediate of electron carrier or various pathways metabolisms.Except its nutritive value, these compounds also have great industrial value as pigment, antioxidant and catalyzer or other processing aids.(for the commentary of these compound structures, activity and industrial application, referring to for example, Ullmann ' s Encyclopedia of Industrial Chemistry, " Vitamins " vol.A27, p.443-613 VCH:Weinheim 1996.) " VITAMIN " this speech is well known in the art, but having comprised that the organism normal function is required again can not self synthetic nutrient substance.VITAMIN can comprise cofactor and dietetic product compound.Term " cofactor " has comprised and has carried out the active required non-protein compound of normal enzyme.These compounds can be inorganic or organic; Cofactor molecule of the present invention is preferably organic." dietetic product " this speech has comprised plant and animal, particularly human body beneficial's dietary supplement.The example of these molecules is VITAMIN, antioxidant and some lipid (for example many saturated fatty acids).

The organism that can produce these molecules, for example biosynthesizing of these molecules in the bacterium, major part identified (Ullman ' s Encyclopedia of Industrial Chemistry, " Vitamins " vol.A27, p.443-613, VCH:Weinheim, 1996; Michal, G. (1999) BiochemicalPathways:An Atlas of Biochemistry and Molecular Biology, John Wiley ﹠amp; Sons; Ong, A.S., Niki, E.﹠amp; 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)

VitB1 (VITMAIN B1) is connected generation with thiazole through chemistry by pyrimidine.Riboflavin (Wei ShengsuB2) is synthetic by 5 '-triphosphoric acid guanosine-and 5 '-ribose phosphoric acid.Riboflavin is used for synthetic vitamin B2 phosphate (FMN) and flavin adenine dinucleotide (FAD) successively.The one group of compound (for example, pyridoxol, Pyridoxylamine, 5 '-pyridoxal phosphate, and commercial pyridoxal hydrochloride) that is collectively referred to as " vitamin B6 " all is the derivative of common structure unit 5-hydroxyl-6-picoline.Pantothenate (pantothenic acid, (R)-(+)-N-(2,4-dihydroxyl-3,3-dimethyl-1-oxo butyl)-Beta-alanine) can obtain by chemosynthesis or fermentation.The biosynthetic final step of pantothenate comprises the Beta-alanine of ATP driving and the condensation of pantoic acid.Be responsible for changing into pantoic acid and Beta-alanine enzyme, and the enzyme that is condensed into pantothenate all is known.The metabolic activity form of pantothenate is a coenzyme A, and its biosynthetic process is 5 enzymatic steps.Pantothenate, 5 '-pyridoxal phosphate, halfcystine and ATP are the precursors of coenzyme A.These enzymes are the formation of catalysis pantothenate not only, also catalysis (R)-pantoic acid, (R)-pantolacton, (R)-generation of panthenol (pro-vitamin B5) pantetheine (and derivative).

In microorganism, study very in detail to the biosynthesizing of vitamin H, and related several genes are identified by precursor molecule pimeloyl coenzyme A.A lot of corresponding proteins matter also are found and have participated in the synthetic of iron bunch (Fe-cluster), and are nifS family protein members.Thioctic Acid is used as coenzyme from sad in energy metabolism, can become the part of pyruvate dehydrogenase complex and ketoglurate dehydrogenase mixture.Folate is the derivative of one group of folic acid, successively from L-L-glutamic acid, para-amino benzoic acid and 6-methylpterin.Originate in the folic acid of metabolism intermediate 5 '-triphosphoric acid guanine (GTP), L-L-glutamic acid and para-amino benzoic acid and the biosynthesizing of derivative thereof, detailed research is arranged in certain micro-organisms.

It is the chemical substance of feature that corrinoid (cobalami for example, and particularly vitamin B12) and porphyrin all belong to the tetrapyrrole ring body.The biosynthesizing of vitamin B12 is such complexity, to such an extent as to also thoroughly do not understand its feature, but many enzymes that relate to and substrate are known now.Nicotinic acid (nicotinate) and nicotine are derivatives at the bottom of the pyridine, also are known as " niacin usp ".Niacin usp is the precursor of important coenzyme NAD (Reduced nicotinamide-adenine dinucleotide) and NADP (Triphosphopyridine nucleotide, reduced) and reduction form thereof.

Although some such compound also can be with extensive microorganism culturing production, for example riboflavin, vitamin B6, pantothenic acid and vitamin H, these compounds of scale operation largely also depend on the acellular chemical system.Has only vitamin B12, because its synthetic complicacy can only be used fermentative production.In vitro method needs considerable material and time to drop into, and often costs a lot of money.

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

Purine and pyrimidine metabolic gene and corresponding proteins matter thereof are the important target compounds of tumor disease therapeutic and treatment of viral infections.Term " purine " and " pyrimidine " have comprised the nitrogenous base of forming as nucleic acid, coenzyme and Nucleotide.Term " Nucleotide " comprises the nucleic acid molecule basic structural unit, and (for RNA, this pentose is a ribose to nucleic acid molecule by nitrogenous base, pentose; For DNA, this pentose is a ribodesose) and the phosphoric acid composition.Term " nucleosides " has comprised the molecule as nucleotide precursor, but the phosphoric acid part that lacks Nucleotide and had.By suppressing the biosynthesizing of these molecules, perhaps suppress to be moving that synthetic nucleic acid molecule carries out, may suppress the synthetic of RNA and DNA; Mode by directed tumour cell suppresses this activity, and tumour cell division and the energy that duplicates may be inhibited.In addition, the Nucleotide that has is not used in formation nucleic acid, but as energy storage (for example AMP) or coenzyme (for example FAD and NAD).

Some publication has been described by influencing the metabolism of purine and/or pyrimidine, these chemical substances as the use of these medical science indications (for example, 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).Relate to the research of purine and pyrimidine metabolic enzyme, concentrate on above the operable new drug development, for example, 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).Yet, purine and pyrimidine bases, nucleosides and Nucleotide also have other effect: as the biosynthetic intermediate of many fine chemistry materials (for example, VitB1, S-adenosylmethionine, folic acid, perhaps riboflavin), as cellular energy carrier (for example ATP or GTP), and as chemical substance itself, usually as flavour enhancer (for example IMP or GMP) or several medical use (referring to, for example, Kuninaka, A. (1996) Nucleotidesand Related Compounds in Biotechnology vol.6, Rehm et al., eds.VCH:Weinheim,, p.561-612).Equally, relate to the enzyme of purine, pyrimidine, nucleosides or nucleotide metabolism, day by day become the effect target of developing as the chemical substance of protecting farm crop, these chemical substances comprise mycocide, weedicide and sterilant.

In the bacterium metabolism of these compounds have feature (comment referring to, Zalkin for example, 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 ", Chapter 8 in:BiochemicalPathways:An Atlas of Biochemistry and Molecular Biology, Wiley:New York).Purine metabolism is an emphasis research topic always, and it is that the cell normal function is necessary.Purine metabolism impaired in the higher animal can cause serious disease, for example gout.Purine nucleotides is synthetic by 5 '-ribose phosphoric acid, pass through series of steps, through intermediate 5 '-phosphoric acid inosine (IMP), finally produce 5 '-single phosphoric acid guanine (GMP) and 5 '-single Vitamin B4 (AMP), and form the triphosphoric acid form that is used as Nucleotide by them.Also as energy storage, it is degraded to, and various biological process provides energy to these compounds in the cell.The biosynthesizing of pyrimidine is by formed 5 '-phosphoric acid uridine (UMP) by 5 '-ribose phosphoric acid.Next UMP is transformed into 5 '-triphosphoric acid cytosine(Cyt) (CTP).The deoxidation form of all these Nucleotide all produces through a step reduction reaction, by the bisphosphate ribose form of the Nucleotide bisphosphate ribodesose form to Nucleotide.Once phosphorylation, these molecules just can participate in the synthetic of DNA.

D. the metabolism of trehalose and purposes

Trehalose comprises two glucose molecules, and by α, α-1,1 connects.Usually in food industries, be used as sweetener, desiccating food products or frozen product additive, and in the middle of the beverage.And, it also be applied in pharmacy, makeup and biotechnology industry (referring to, Nishimoto et al. for example, (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 a lot of microorganisms can produce trehalose, and with its natural being discharged on every side in the substratum, and the method for knowing on can use technology is therefrom collected.

II. phosphoenolpyruvic acid: carbohydrate phosphotransferase system

Quick growth and the division of cell in substratum depends on cellular uptake and the degree of utilizing the high energy molecule, for example glucose or other carbohydrates to a great extent.Have different transport proteins, they are transported to different carbohydrates in the cell.Have carbohydrate translocator matter, for example transport glucose, fructose, seminose, semi-lactosi, ribose, sorbose, ribulose, lactose, maltose, sucrose, perhaps raffinose also has the translocator matter of starch and cellulose degradation product.Other movement systems are responsible for importing alcohol (for example methyl alcohol or ethanol), alkane, lipid acid, and the organic acid as acetate or lactic acid.In bacterium, by various mechanism, carbohydrate can be transported into cell through cytolemma.Except with the cotransporting of proton, one of carbohydrate capture process of the most normal use is a phosphoenolpyruvic acid: carbohydrate phosphotransferase system (PTS).This system is the transhipment (following phosphorylation) of catalysis carbohydrate and hexitol not only, but also regulates the cellular metabolism that is adapted to carbohydrate validity.This PTS system only is present in the bacterium, and does not appear in archeobacteria and the eukaryotic cell.

Function aspects, PTS system comprise two kinds of cytoplasm proteins, enzyme I and HPr, and the special integration of the carbohydrate of indefinite number and peripheral membrane protein transhipment complex body (every kind all is called as and has carbohydrate special target " enzyme II ", for example " enzyme II down ^Glu" expression is in conjunction with the enzyme II complex body of glucose).The known enzyme II special to monose, disaccharides or trisaccharide similarly is to glucose, fructose, seminose, semi-lactosi, ribose, sorbose, ribulose, lactose, maltose, sucrose or raffinose.Enzyme I is transferred to phosphate carrier albumen HPr to phosphate group from phosphoenolpyruvic acid (PEP).HPr is transferred to different enzyme II transhipment complex bodys to phosphate group more then.Though the aminoacid sequence of enzyme I and HPr is very similar in all bacteriums, PTS shifts body can be divided into the incoherent several families of structure.In addition, the number of these genes also has nothing in common with each other in different bacteriums with homology.38 different pts proteins of bacillus coli gene group coding, wherein 33 is to belong to 22 different subunits that shift body.Mycoplasma genitalium (M.genitalium) genome contains each one of enzyme I and HPr gene, and has only two PTS to shift the body genes.T.palladium and chlamydia trachomatis (C.trachomatis) contain enzyme I and HPr similar protein gene, but do not have PTS to shift the body gene.

All PTS shift bodies and comprise 3 functional units, IIA, protein subunit (for example, the IIA among IIB and the IIC, they or complex body ^GlcIICB ^Glc) or structural region (for example, the IICBA of single polypeptide chain ^GlcNAc).IIA and IIB are delivered to the carbohydrate of being transported to phosphate group successively from HPr.IIC contains the carbohydrate binding site, and crosses over inner membrance 6 or 8 times.Carbohydrate shifts and the instantaneous phosphorylation in IIB zone is coupling.Enzyme I, HPr and IIA are in its histidine residues generation phosphorylation, and the IIB subunit is in histidine residues or cysteine residues generation phosphorylation, this depends on related specific transporter.The phosphorylation of input back carbohydrate has such advantage, promptly can stop carbohydrate to spread cytolemma and gets back in the substratum, because charged phosphate group can not pass the hydrophobic core of cytolemma.

Some pts protein also plays a significant role in the signal conduction in cell except active carbohydrate transport function.These subunits are regulated their object by allosteric or phosphorylation.Their adjusting activity changes along with phosphorylation degree (for example, the ratio of non-phosphorylating form and phosphorylation form), and the latter relies on the ratio of phosphorylation along with sugar dependence dephosphorylation and phosphoenolpyruvic acid again and changes.The example of regulating in the cell of this pts protein in the intestinal bacteria comprises, passes through IIA ^GlcDephosphorylation suppresses glycerol kinase, and by its phosphorylation form activated adenyl cyclase.And express by the reversible phosphorylation regulatory gene of transcribing anti-terminator in the HPr of some transporter and IIB zone in these microorganisms.In gram positive bacterium, the activity of HPr is regulated by special serine kinase of HPr and Phosphoric acid esterase.For example, the HPr of Serine-46 phosphorylation, its function is the co-repressor that suppresses sub-CcpA as transcribing.At last, find that unphosphorylated enzyme I suppresses the sensor kinase c heA in the bacterium chemotactic device, this kinases provides between the system of bacterium carbohydrate binding transport system and the motion of control bacterium and has contacted directly (Sonenshein, 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 present invention and method

The present invention is to be based upon on the basis of finding the recruit to small part, it is called PTS nucleic acid and protein molecule herein, they to the high-energy carbon molecule (for example participate in Corynebacterium glutamicum, glucose, fructose, sucrose) picked-up, and also can participate in these microorganisms signal transduction path in the one or more of cells.In one embodiment, the PTS molecule is exercised the function that the high-energy carbon molecule is changed over to cell, the energy that these molecular degradations provided in cell can be the disadvantageous biochemical reaction energy supply of energy, and their degraded product can be as the intermediate or the precursor of a lot of other pathways metabolisms.In another embodiment, the PTS molecule can participate in signal transduction path, the wherein existence of PTS molecular modification form (for example, phosphorylation PTS molecule) in the one or more of cells, can participate in the signal transduction cascade reaction, this cascade reaction is regulated one or more cell processes.In a preferred embodiment, the activity of PTS molecule of the present invention is for influential with the required fine chemistry material of this microorganisms producing.In an especially preferred embodiment, the activity of PTS molecule of the present invention is conditioned, and makes that output, production and the production efficiency of one or more fine chemistry materials has also obtained adjusting in the Corynebacterium glutamicum.

Term " pts protein " or " PTS polypeptide " have comprised those participations and (for example absorbed one or more high energy compounds to cell interior from the substratum of extracellular, monose, disaccharides or oligosaccharides similarly are fructose, seminose, sucrose, glucose, raffinose, semi-lactosi, ribose, lactose, maltose and sorbose) protein.These pts proteins also can participate in signal transduction path in the one or more of cells, but for example not only are confined to, and those are controlled different carbohydrates and absorb the into approach of cell.The example of pts protein comprises that sequence number is the protein of the PTS genes encoding of odd number in sequence table by being listed in for those.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 nucleotide sequence " have comprised the nucleotide sequence of coding pts protein, and the latter comprises 5 ' and 3 ' sequence area of coding region and corresponding untranslated.The example of PTS gene comprises that those are listed in the gene in the table 1.Term " production " or " productivity " are well known in the art, have comprised in preset time and given fermentation volume the concentration of tunning (for example, required fine chemistry material) (for example, per hour every liter of kilogram product).Term " production efficiency " has comprised, and reach the specific required time (for example, needing how long just to make cell reach specific fine chemistry material) of production level.Term " income " " product/carbon income " is well known in the art, has comprised the efficient that carbon source is changed into product (for example fine chemistry material).For example, often write every kilogram of carbon source of kilogram product.By improving the income or the production of compound, can increase the quantity that reclaims molecule, perhaps be increased in the quantity of giving the useful recovery molecule of this compound in the culture of determined number in preset time.Term " biosynthesizing " or " biosynthetic pathway " are well known in the art, have comprised in cell, may be multisteps and be the processes of highly regulating and control from middle compound process, synthetic compound, particularly organic compound.Term " degraded " or one " degradation pathway " are well known in the art, have comprised in cell, through being multistep and being the process of highly regulating and control, compound, preferably organic compound is decomposed into degraded product (molecule that generally speaking, be littler or complicacy is littler).Term " metabolism " is well known in the art, has comprised the whole of the biochemical reaction that taken place in the organism.Thereby the metabolism of special compound (for example, similarly being the such amino acid metabolism of glycine) comprises that all biological relevant with this compound in the cell is synthetic, modification and degradation pathway.Term " transhipment " and " input " be well known in the art, comprised the facilitation that one or more compounds pass cytolemma and moved, and these compounds can not pass cytolemma by other mode.

In another embodiment, PTS molecule of the present invention can be regulated in the microorganism, and for example in the Corynebacterium glutamicum, required compound is the generation of fine chemistry material for example.Use the genetic recombination technology can operate one or more PTS molecules of the present invention, thereby regulate its activity.For example, a kind of protein that participates in the glucose input of PTS mediation can be conditioned, and makes its active optimization, and the PTS system that makes the glucose input, and the glucose that can transport greater amt is in cell.Because glucose molecule not only can be as energy to promote the unfavorable biochemical reaction of energy, for example fine chemistry material biosynthesizing, and can be used as the precursor and the intermediate of many biological fine chemistry material biosynthetic pathways (for example, from 3-phoshoglyceric acid synthetic Serine).In each example, perhaps provide by increasing the required energy of production generation, perhaps produce the required compound supply of generation by increasing, can increase the ultimate production or the production efficiency of these required fine chemistry materials.

In addition, a lot of known pts proteins play a crucial role in the signal transduction path in cell, and these approach are regulated and kept cellular metabolism and the carbohydrate picked-up that carbon source is supplied with.For example, in the known cell 1, the increase of 6-hexose diphosphate (compound that produces in the glycolysis-) level can cause the phosphorylation of HPr serine residue, thus stop this protein in any PTS carbohydrate transport process as the phosphate group donor.Make that by mutagenesis HPr its serine residue can not be by phosphorylation, activation HPr that can composition, thereby the carbohydrate in the increase transporte to cells can be guaranteed to be used in the cell the required more energy of one or more required fine chemistry material biosynthesizing then and store and intermediate/precursor molecule.

Isolating nucleotide sequence of the present invention is included in the genome of Corynebacterium glutamicum strain, and this bacterial strain can be obtained by American type culture collection, preserving number ATCC 13032.Isolating Corynebacterium glutamicum PTS DNA nucleotide sequence, and the Corynebacterium glutamicum pts protein aminoacid sequence of prediction are listed with odd serial numbers and even number sequence number respectively in sequence table.

Carried out Computer Analysis, and with the classification of these nucleotide sequences and/or be accredited as the sequence of encoding metabolic pathway proteins matter.

The present invention is also relevant with such protein, and this proteinic aminoacid sequence and aminoacid sequence of the present invention have sufficient homology (for example, the sequence of even number sequence number in the sequence table).Such as used herein, have the protein that the aminoacid sequence of abundant homology is arranged with the aminoacid sequence of picking out, with the aminoacid sequence of picking out, the amino acid complete sequence of for example picking out has about at least 50% homology.The aminoacid sequence that has and pick out has the protein of the aminoacid sequence of very big homology, also can at least approximately 50-60% be arranged with the aminoacid sequence of picking out, at least about 60% homology is preferably arranged, and preferred have about at least 70%, 80%, 90% homology, most preferred have about at least 95%, 96%, 97%, 98%, 99% or higher homology.

Pts protein of the present invention or its biologically-active moiety or its fragment, can participate in transporting similarly is that the such high energy carbon-containing molecules of glucose enters Corynebacterium glutamicum, perhaps participate in signal conduction in the cell in this microorganism, perhaps have one or more activity of listing in the table 1.

Following each several part has been described all respects of the present invention in further detail:

A. isolated nucleic acid molecule

One aspect of the present invention relates to the nucleic acid molecule of separated coding PTS polypeptide or its biologically-active moiety, and enough as the nucleic acid molecule fragment of hybridization probe or primer, these fragments are used to identify or the nucleic acid (for example PTS DNA) of amplification coding PTS.Such as used herein, the meaning of term " nucleic acid molecule " is to comprise dna molecular (for example, cDNA or genomic dna) and RNA molecule (for example mRNA), and by the DNA or the RNA analogue of nucleotide analog deposits yields.This term also comprises and is positioned at genes encoding zone 3 ' and 5 ' terminal non-translated sequence: at least 100 Nucleotide of coding region 5 ' terminal upstream sequence and at least 20 Nucleotide of genes encoding zone 3 ' terminal downstream sequence.Nucleic acid molecule can be strand or double-stranded, but double-stranded DNA preferably." isolating " nucleic acid molecule is meant those and is present in the nucleic acid molecule that other nucleic acid molecule in the nucleic acid natural origin are separated from each other.Preferably, " isolating " nucleic acid does not contain the natural sequence (for example, being positioned at nucleic acid 5 ' and 3 ' terminal sequence) that is positioned at organism genomic dna amplifying nucleic acid both sides, and nucleic acid obtains from this organism.For example, in various embodiments, isolating PTS nucleic acid molecule can contain and be less than about 5kb, 4kb, 3kb, 2kb, 1kb, the nucleotide sequence of 0.5kb or 0.1kb, the natural both sides that are positioned at the cell genomic dna nucleic acid molecule of this sequence, nucleic acid is exactly to obtain from these cells (for example, Corynebacterium glutamicum cell).In addition, " isolating " nucleic acid molecule, for example dna molecular can be substantially devoid of other cellular materials or substratum when producing with recombinant technology, can not contain precursor or other chemical substances when chemosynthesis.

Nucleic acid molecule of the present invention, the nucleotide sequence of odd serial numbers in the sequence table for example, perhaps its part can separate obtaining by standard molecular biological technique with sequence information provided herein.For example, Corynebacterium glutamicum PTS DNA can be from the Corynebacterium glutamicum library, use in the sequence table in the odd serial numbers sequence whole or its part of a sequence as hybridization probe, and the standard hybridization technique (similarly is to be described in Sambrook, J. for example,, Fritsh, E.F., and Maniatis, T.Molecular Cloning:A Laboratory Manual.2 ^Nd, ed.Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY is in 1989) and separation obtains.In addition, (for example comprise a nucleotide sequence of the present invention, the nucleotide sequence of odd serial numbers in the sequence table) whole or a part of nucleic acid molecule, can pass through the polymerase chain reaction, use is based on the oligonucleotide primer of this sequences Design, separate and (for example obtain, (for example comprise a nucleotide sequence of the present invention, the nucleotide sequence of odd serial numbers in the sequence table) whole or a part of nucleic acid molecule, can pass through the polymerase chain reaction, use separates obtaining based on the oligonucleotide primer of this identical sequence design).For example, mRNA can (for example obtain from the normal endothelial cell separation, use the guanidine thiocyanate extracting method among Chirgwin et al. (1979) the Biochemistry 18:5294-5299), DNA can be by reversed transcriptive enzyme (for example, Gibco/BRL, the Moloney MLV reversed transcriptive enzyme that Bethesda, MD provide; Perhaps SeikagakuAmerica, Inc., the AMV reversed transcriptive enzyme that St.Peterburg, FL provide) preparation.Be polymerase chain reaction synthetic oligonucleotide primer, can be based on a nucleotide sequence design of listing in the sequence table.Nucleic acid of the present invention can use cDNA or make template as the genomic dna that another kind is selected, and suitable oligonucleotide primer increases according to Standard PC R amplification technique.The nucleic acid that amplifies like this can be cloned in the suitable carriers, and distinguishes its feature with dna sequence analysis.In addition,, can prepare, for example use automatic dna synthesizer with the standard synthetic technology with the corresponding oligonucleotide of PTS nucleotide sequence.

In a preferred embodiment, isolating nucleic acid molecule of the present invention comprises a nucleotide sequence of listing in the sequence table.Nucleotide sequence of the present invention, as in sequence table, list those, be consistent with Corynebacterium glutamicum PTS DNA of the present invention.These DNA comprise the sequence (i.e. " coding region " is presented at odd serial numbers in every sequence table: in the sequence) of the pts protein of encoding, and 5 ' non-coding sequence and 3 ' non-coding sequence, are also shown in odd serial numbers in every sequence table: in the sequence.Select as another kind, nucleic acid molecule can only comprise the coding region of sequence table amplifying nucleic acid sequence.

For the purpose of this application, be appreciated that every nucleic acid and the aminoacid sequence listed in the sequence table, a RXA who is used to discern is all arranged, RXN, RXS or RXC coding are indicated " RXA ", " RXN ", " RXS ", perhaps there are 5 numerals (that is, RXA01503, RXN01299 in " RXC " back, RXS00315, perhaps RXC00953).Every nucleotide sequence comprises three parts at most: 5 ' upstream region, coding region, downstream area.Trizonal each part is all used identical RXA, RXN, and RXS, perhaps the RXC numbering is determined to obscure with elimination.So narration " sequence of odd number coding in the sequence table " is meant any nucleotide sequence in the sequence table, these sequences also can be with they different RXA, RXN, RXS, perhaps RXC numbering differentiation mutually.The coding region of every this sequence all is translated into amino acid sequence corresponding, and these sequences also are listed in the sequence table, for following corresponding nucleic sequence even number sequence number afterwards closely:.For example, the coding region of RXA02229 is listed in SEQ ID NO:1, and its amino acid sequence coded is listed in SEQ ID NO:2.Sequence of nucleic acid molecules of the present invention, with its amino acids coding molecule, with identical RXA, RXN, RXS, perhaps the expression of RXC numbering makes them connect each other easily.For example, be appointed as RXA01503, RXN01299, the aminoacid sequence of RXS00315 and RXC00953 is respectively RXA01503, RXN01299, the translation in RXS00315 and the nucleotide sequence coded zone of RXC00953 nucleic acid molecule.RXA of the present invention, RXN, the correspondence between RXS and RXC Nucleotide and the aminoacid sequence, and their appointed sequence number column are in table 1.For example, similarly be that the nucleotide sequence RXN01299 that is listed in the table 1 is SEQ ID NO:7, amino acid sequence corresponding is SEQ ID NO:8.

Several gene of the present invention is " gene that F-indicates ".The gene that F-indicates comprises that those are listed in the table 1 and at RXA, RXN, and RXS has the gene of " F " before perhaps RXC indicates.For example, SEQ ID NO:3 is as expression in table 1, be designated as " F RXA00315 ", gene that F-indicates exactly, same also have SEQ ID NO:9,11 and 13 (be marked as " FRXA01229 ", FRXA01883 in the table 1 respectively " and " F RXA01889 ").

In one embodiment, nucleic acid molecule of the present invention does not comprise those Corynebacterium glutamicum molecules that are compiled in the table 2.For the dapD gene, the sequence of this gene is published in Wehrmann, A., et al. (1998) J.Bacteriol.180 (12): 3159-3165.Yet the version that the resulting ratio of the present application person is delivered is long a lot.It is said that the version of delivering has used wrong codon in fact, and therefore only showed the part of true coding region.

In another preferred embodiment, isolating nucleic acid molecule of the present invention, sequence) or the nucleic acid molecule of the complementary molecule of its part comprising those is nucleotide sequence of the present invention (for example, odd serial numbers in the sequence table:.With a nucleotide sequence complementary of the present invention nucleic acid molecule, be meant that a nucleotide sequence listing in this molecule and the sequence table (for example, odd serial numbers: sequence) fully complementary, so it can with a nucleotide sequence hybridization of the present invention, thereby form stable duplex.

Equally in another preferred embodiment, isolating nucleic acid molecule of the present invention comprises such nucleotide sequence, and this sequence and nucleotide sequence of the present invention are (for example, sequence) or its part odd serial numbers in the sequence table:, have about at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% homology preferably has about at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% homology, preferred have about at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90%, perhaps 91%, 92%, 93%, 94%, and even preferredly have about at least 95%, 96%, 97%, 98%, 99% or higher homology.More than quote scope (for example, 70-90% consistence or 80-95% consistence) intermediary scope and consistence value, be also contained among the present invention.For example, comprised such consistence value scope, these scopes are combinations of the above-cited upper limit and/or lower value.In another kind of embodiment preferred, isolated nucleic acid molecule of the present invention comprises such nucleotide sequence, this sequence can with a nucleotide sequence of the present invention (for example, odd serial numbers in the sequence table: sequence) or its part hybridize, for example, hybridize under stringent condition.

In addition, nucleic acid molecule of the present invention may only comprise the part in odd serial numbers sequence encoding zone in the sequence table, for example, and can be as the fragment of probe or primer, the fragment of the pts protein biologically-active moiety of perhaps encoding.The nucleotide sequence that goes out by Corynebacterium glutamicum PTS gene clone, allow and produce probe and primer, these probes and primer design are the PTS homologues that is used for identifying and/or cloning other cell types or other biological body, and the PTS homologue in other excellent bacillus or the affinity species.Probe/primer typically comprises the oligonucleotide of suitable purifying.Oligonucleotide typically comprises the zone of such one section nucleotide sequence, this zone is under stringent hybridization condition, with nucleotide sequence of the present invention (for example, odd serial numbers sequence in the sequence table) sense strand, the antisense sequences of these sequences, perhaps about at least 12 of its naturally occurring mutant, preferred about 25, preferred about 40,50, perhaps 75 continuous nucleotides hybridization.Based on the primer of nucleotide sequence of the present invention, can be used to clone the PCR reaction of PTS homologue.Based on the probe of PTS nucleotide sequence, can be used to survey transcribing or genome sequence of identical or homologous protein.In a preferred embodiment, probe comprises the other labelling groups that adheres to especially, and for example labelling groups can be the cofactor of radio isotope, fluorescent chemicals, enzyme or enzyme.This probe can be as the part of diagnostic test kits, this test kit is used to identify the cell of false demonstration pts protein, similarly be by measuring the level of PTS coding nucleic acid in the sample cell, for example, detect the level of PTS mRNA, perhaps measure genome PTS gene whether sudden change or disappearance have taken place.

In one embodiment, a kind of protein of nucleic acid molecule encoding of the present invention or its part, the aminoacid sequence of this protein or its part and aminoacid sequence of the present invention are (for example, even number sequence number sequence in the sequence table) sufficient homology is arranged, thereby make this protein or its part have the ability also can participate in signal transduction path in the one or more of cells to high-energy carbon molecule (for example glucose) input Corynebacterium glutamicum.Such as used herein, term " homology fully " is meant the aminoacid sequence of protein or its part, contain minimal number with consensus amino acid sequence of the present invention or of equal value (for example, have to sequence table even number sequence number sequence in the amino-acid residue of the similar side chain of amino-acid residue) amino-acid residue, thereby make this protein or its part, can also can participate in signal transduction path in the one or more of cells in this microorganism similarly being the such high-energy carbon molecule input Corynebacterium glutamicum of glucose.The protein member of this pathways metabolism, as described herein, its function is similarly being the such high-energy carbon molecule input Corynebacterium glutamicum of glucose, also can participate in signal transduction path in the one or more of cells in this microorganism.This active example has also been described here.Thereby, " function of pts protein " has contribution for the repertoire and/or the adjusting of one or more of carbohydrate transporting pathway based on phosphoenolpyruvic acid, and/or the direct or indirect output to one or more fine chemistry materials, production and/or production efficiency have contribution.The active example of pts protein is listed in table 1.

In another embodiment, protein and whole aminoacid sequence of the present invention have at least approximately homology of 50-60%, the approximately homology of 60-70% is preferably arranged at least, and preferred have at least approximately 70-80%, a 80-90%, the homology of 90-95%, most preferred have about at least 96%, 97%, 98%, 99% or higher homology (for example, even number sequence number in the sequence table: sequence).

The proteinic part of PTS nucleic acid molecule encoding of the present invention, the preferably biologically-active moiety of pts protein.Such as used herein, the meaning of term " biologically-active moiety of pts protein " is to comprise the such part of pts protein, structural domain/primitive for example, this part can perhaps participate in signal transduction path in the one or more of cells in this microorganism similarly being the such high-energy carbon molecule input Corynebacterium glutamicum of glucose.Can carry out a kind of enzyme activity assay, to determine whether pts protein or its biologically-active moiety have participated in perhaps having participated in similarly be the such high-energy carbon molecule input Corynebacterium glutamicum of glucose in signal transduction path in the one or more of cells in this microorganism.The person knows this analytical procedure for being familiar with the routine techniques, in the example 8 of example detailed description is arranged.

The extra nucleic acid fragment of coding pts protein biologically-active moiety, can prepare by the following method, (for example separate aminoacid sequence of the present invention, even number sequence number in the sequence table: part sequence), the encoding part of expression pts protein or polypeptide (for example, pass through in-vitro recombination expression), and the activity of estimation pts protein or peptide coding part.

Because the degeneracy of genetic codon, and can encode thus and obtain and the identical pts protein of nucleotide sequence encoding protein matter of the present invention, so further comprising, the present invention is different from nucleotide sequence of the present invention (for example, odd serial numbers in the sequence table: the sequence) nucleic acid molecule of (with its part).In another embodiment, isolating nucleic acid molecule of the present invention has such nucleotide sequence, and this sequence encoding has the protein of the aminoacid sequence of listing in the sequence table (for example, even number sequence number :).Equally in another embodiment, the Corynebacterium glutamicum protein of nucleic acid molecule encoding total length of the present invention, this protein and aminoacid sequence of the present invention are (by odd serial numbers in the sequence table: the open reading frame coding) sufficient homology is arranged.

In one embodiment, technical known sequences before sequence of the present invention and not meaning that comprises, for example those are listed in the just effective Genbank sequence before the present invention in table 2 or the table 4, and person is understandable for being familiar with the routine techniques for this.In one embodiment, the present invention comprises such nucleotide sequence and aminoacid sequence, this sequence and nucleotide sequence of the present invention and aminoacid sequence have the consistence of certain percentage, this per-cent is greater than the per-cent of technical known sequences (the Genbank sequence of for example, listing in table 2 or the table 4 (the perhaps protein of this sequence encoding)) with nucleotide sequence of the present invention and consensus amino acid sequence.For example, the present invention comprise with the nucleotides sequence that is marked as RXA01503 (SEQ ID NO:5) show greater than and/or at least 44% conforming nucleotide sequence, with the nucleotides sequence that is marked as RXA00951 (SEQ ID NO:15) show greater than and/or at least 41% conforming nucleotide sequence, and with the nucleotides sequence that is marked as RXA01300 (SEQ ID NO:21) show greater than and/or at least 38% conforming nucleotide sequence.The person that is familiar with the routine techniques, calculate the per-cent consistence by 3 GPA-that meet the highest that provide for each particular sequence that list in the look-up table 4, and deduct the highest GPA-in 100 percent through associating and calculate the per-cent consistence, can calculate the conforming low side thresholding of any particular sequence per-cent of the present invention.The person also can recognize to be familiar with the routine techniques, and its per-cent consistence is greater than the low side thresholding that so calculates (for example, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably about at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, preferred about at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90%, perhaps 91%, 92%, 93%, 94%, and even preferred about at least 95%, 96%, 97%, 98%, 99% or higher consistence) nucleic acid and aminoacid sequence, also be included among the present invention.

The person can recognize to be familiar with the routine techniques, except the Corynebacterium glutamicum PTS nucleotide sequence of listing with odd serial numbers in sequence table, the dna polymorphism that causes the pts protein aminoacid sequence to change can exist in certain colony (for example Corynebacterium glutamicum colony).This PTS Gene Polymorphisms can exist in the Different Individual of a colony owing to the variation of natural condition.Such as used herein, term " gene " and " recombination " are meant the nucleic acid molecule of the open reading frame that contains the pts protein of encoding, and preferred pts protein is the Corynebacterium glutamicum pts protein.The variation of this natural condition typically can cause the variation of PTS gene nucleotide series 1-5%.Any and all cause, and do not change the pts protein functionally active owing to the variation of natural condition, the variation of this Nucleotide, and the amino acid whose polymorphism of the PTS that causes all belong within the scope of the invention.

The nucleic acid molecule of corresponding natural variant, non-glutamic acid rod bacillus homologue with Corynebacterium glutamicum PTS DNA of the present invention, can be based on the homology of disclosed herein they and Corynebacterium glutamicum PTS nucleic acid molecule, use Corynebacterium glutamicum DNA or its part as hybridization probe, separation obtains according to the standard hybridization technique under stringent hybridization condition.Therefore, in another embodiment, the length of isolating nucleic acid molecule of the present invention has 15 Nucleotide at least, under stringent condition with contain the ordered list odd serial numbers: the making nucleic acid molecular hybridization of nucleotide sequence.In other embodiments, the length of nucleic acid molecule has 30,50 at least, and 100,250 or more a plurality of Nucleotide.Such as used herein, the meaning of term " hybridize under stringent condition " is to describe the such hybridization and the condition of cleaning, has the nucleotide sequence of at least 60% homology to keep typical hybridization each other under this condition each other.Preferably, this condition be have between the sequence about at least 65%, preferred have about at least 70%, and even preferredly have about at least 75 or higher homology, keep typical hybridization each other.The person is known to this stringent condition for being familiar with the routine techniques, can be at Ausubel et al., and Current Protocols in Molecular Biology, JohnWiley ﹠amp; Sons, N.Y. (1989) finds among the 6.3.1-6.3.6.Preferably a kind of but be not that the stringent hybridization condition of restriction is, in 6X sodium chloride/sodium citrate (SSC), hybridize for about 45 ℃, use 0.2X SSC then, 0.1%SDS is 50-65 ℃ of cleaning once or repeatedly.Preferably, isolating nucleic acid molecule of the present invention with nucleotide sequence hybridization of the present invention, is equivalent to obtain naturally occurring nucleic acid molecule under stringent hybridization condition.Such as used herein, " naturally occurring " nucleic acid molecule is meant RNA or the dna molecular with the nucleotide sequence (for example, coding natural protein) that exists in the nature.In one embodiment, the natural Corynebacterium glutamicum pts protein of nucleic acid encoding.

The person can further recognize to be familiar with the routine techniques, the naturally occurring PTS sequence variants that in colony, exists, can be by sudden change change introducing in the nucleotide sequence of the present invention, thus the change of the aminoacid sequence of the pts protein that causes being encoded, and do not change the function of pts protein.For example, can be in nucleotide sequence of the present invention, carry out causing the Nucleotide of the aminoacid replacement of " nonessential " amino-acid residue to replace." nonessential " amino-acid residue is meant such residue, this residue can be at the wild-type sequence of pts protein (for example, even number sequence number in the sequence table: change sequence), and do not change the activity of pts protein, and " essential " amino-acid residue is that the pts protein activity is necessary.Yet other amino-acid residues (for example, those are nonconservative or be semiconservative amino-acid residue in the PTS active structure domain) may be optional for activity, therefore also can be changed not changing under the active situation of PTS.

Therefore, another aspect of the present invention relates to the nucleic acid molecule of coding such pts protein, and this pts protein contains the variation to the active nonessential amino-acid residue of PTS.These proteinic aminoacid sequences are different from even number sequence number in the sequence table: sequence, but still keep at least a PTS activity described herein.In one embodiment, isolated nucleic acid molecule comprises the nucleotide sequence of one section coded protein, wherein this proteinic aminoacid sequence and aminoacid sequence of the present invention have about at least 50% homology, and can be similarly being that the such high-energy carbon-containing molecules of glucose is transported into Corynebacterium glutamicum, perhaps participate in signal conduction in the cell in this microorganism, perhaps have one or more activity of listing in the table 1.Preferably, odd serial numbers aminoacid sequence in the protein of nucleic acid molecule encoding and the sequence table, at least approximately the homology of 50-60% is arranged, and preferred and this sequence has at least approximately homology of 60-70%, even preferred and this sequence has at least approximately 70-80%, 80-90%, the homology of 90-95%, aminoacid sequence most preferred and of the present invention has about at least 96%, 97%, 98%, perhaps 99% homology.

In order (for example to determine two seed amino acid sequences, a kind of aminoacid sequence of the present invention and its mutant forms) or the percent homology of two kinds of nucleotide sequences, for optimum purpose relatively, sequence (is for example carried out the sequence contrast, in order to carry out optimum sequence contrast, can in the sequence of a kind of protein or nucleic acid, introduce the gap) with other protein or nucleic acid.The Nucleotide of the amino-acid residue of more corresponding amino acid position or nucleic acid position then.When a sequence (for example, an aminoacid sequence of the present invention) position in by with other sequences (for example, when the mutant forms of aminoacid sequence) identical amino-acid residue in corresponding position or Nucleotide occupy, this molecule is homologous (that is, " consistence " of amino acid or nucleic acid " homology " and amino acid or nucleic acid is identical as used herein) in this position.Per-cent homology between the two sequences is the function (that is # * 100 of the # of % consistence=same position/whole positions) that a same position number is divided equally by sequence.

Isolating and protein sequence of the present invention are (for example, even number sequence number in the sequence table: the sequence) nucleic acid molecule of homologous coding pts protein matter, can replace by in nucleotide sequence of the present invention, introducing one or more Nucleotide, insert, disappearance produce, thereby in coded protein, introduce one or more aminoacid replacement, insertion, disappearance.Can use standard technique, for example site-directed mutagenesis and PCR mediated mutagenesis are introduced sudden change in nucleotide sequence of the present invention.Preferably, Bao Shou aminoacid replacement is that non-essential amino acid residue in one or more expection carries out." conservative aminoacid replacement " is meant that the amino-acid residue that amino-acid residue is had a similar side chain replaces.Have the amino-acid residue family of similar side chain, regulation is arranged technically.These families comprise, amino acid with basic side chain (for example, Methionin, arginine, Histidine), amino acid with acid side-chain (for example, aspartic acid, L-glutamic acid), amino acid with no charge polarity side chain (for example, glycine, aspartic acid, glutamine, Serine, Threonine, tyrosine, halfcystine), amino acid with non-polar sidechain (for example, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), amino acid with β-side chain side chain (for example, Threonine, Xie Ansuan, Isoleucine), and amino acid (for example, tyrosine with fragrance group side chain, phenylalanine, tryptophane, Histidine).Therefore, the non-essential amino acid residue in the pts protein of expection is preferably by other aminoacid replacement in the same side chain family.In addition, in another embodiment, can be in PTS encoding sequence total length or part, introducing sudden change at random for example by saturation mutagenesis, has the PTS activity of PTS active mutant according to evaluation described herein, filters out the mutant that obtains.After the mutagenesis of odd serial numbers nucleotide sequence, the protein that is encoded can be recombinant expressed in a sequence table, protein active also can, for example use analysis described herein (referring to the example 8 of example), determined.

Except the nucleic acid molecule of coding pts protein matter described above, another aspect of the present invention is also relevant with isolating antisense nucleic acid molecule." antisense " nucleic acid comprises " justice is arranged " nucleic acid complementary nucleotide sequence with coded protein, for example with the complementation of double chain DNA molecule coding strand, perhaps with the complementation of mRNA sequence.Therefore, antisense nucleic acid can be connected with phosphorothioate odn is arranged by hydrogen bond.Antisense nucleic acid can with the complementation of whole PTS coding strand, also can be only and its part complementation.In one embodiment, antisense nucleic acid molecule is with " coding region " antisense of nucleotide sequence coded chain of coding pts protein.Term " coding region " is meant the nucleotides sequence column region (for example, whole coding regions of SEQ ID NO.5 (RAX01503) comprise 1 to 249 Nucleotide) that comprises the codon of translating into amino-acid residue.In another embodiment, antisense nucleic acid molecule is with the antisense of nucleotide sequence coded chain of coding PTS.Term " non-coding region " is meant that the coding region both sides do not translate into amino acid whose 5 ' and 3 ' sequence (i.e. 5 ' and 3 ' untranslated zone).

Consider the coding strand sequence (for example, the odd serial numbers sequence of listing in the sequence table) of the coding PTS that announces herein, antisense nucleic acid of the present invention can design according to the base pairing rules of Watson and Crick.Antisense nucleic acid molecule can with whole coding region complementations of PTS mRNA, but be more preferably such oligonucleotide, this oligonucleotide is an antisense with the coding region of PTS mRNA or the part of non-coding region only.For example, antisense oligonucleotide can with near the regional complementarity the PTS mRNA translation initiation position.For example, the length of antisense oligonucleotide can be 5,10,15,20,25,30,35,40, and 45 or 50 Nucleotide.Antisense nucleic acid molecule of the present invention, can use technology on known program, make up by chemosynthesis or enzymatic ligation.Can use naturally occurring Nucleotide or various Nucleotide through modifying, chemosynthesis antisense nucleic acid (for example antisense oligonucleotide), those Nucleotide through modifying, be in order to increase the biologically stable of molecule, perhaps in order to increase antisense nucleic acid and to have between the phosphorothioate odn the double-helical physical stability of formation design, the Nucleotide that for example can use thiophosphoric acid derivative and acridine to replace.The example that can be used for producing the modified Nucleotide of antisense nucleic acid comprises, 5 FU 5 fluorouracil, 5-bromouracil, the 5-chlorouracil, 5-iodouracil, xanthoglobulin, xanthine, the 4-acetylcytosine, 5-(carboxyl hydroxymethyl) uridylic, 5-carboxymethylamino methyl-2-thiouracil, 5-carboxymethylamino 6-Methyl Uracil, dihydrouracil, beta-D-galactosyl inosine, N6-isopentyl VITAMIN B4, the 1-methyl guanine, the 1-methylinosine, 2, the 2-dimethylguanine, the 2-methyladenine, the 2-methyl guanine, 3-methylcystein, 5-methylcytosine, the N6-VITAMIN B4, the 7-methyl guanine, 5-methylamino 6-Methyl Uracil, 5-methoxyl group amino methyl uridylic-2-sulfo-uridylic, beta-D-mannose group queosine, 5 '-methoxyl group carboxyl 6-Methyl Uracil, 5-methoxyuracil, 2-methyl sulfo--N6-isopentyl VITAMIN B4, uridylic-5-contains fluoroacetic acid (V), wybutoxosine, pseudouracil, queosine, 2-sulfo-cytosine(Cyt), 5-methyl-2-sulfo-uridylic, 2-sulfo-uridylic, 4-sulfo-uridylic, methyl uracil, uridylic-5-contains the fluoroacetic acid methyl esters, and uridylic-5-contains fluoroacetic acid (v), 5-methyl-2-sulfo-uridylic, 3-(3-amino-3-N-2-carboxyl propyl group) uridylic, (acp3) w, and 2,6-diaminopurine.In addition, antisense nucleic acid can use the expression vector biosynthesizing, and its amplifying nucleic acid (that is, by the RNA that inserts transcribed nucleic acid, is antisense orientation with respect to the purpose nucleic acid that inserts, with the lower section further narration is arranged) in expression vector by the antisense orientation subclone.

Antisense nucleic acid molecule of the present invention, typically be applied to cell or produce in position, thereby they can or combine with the cell mRNA and/or the genomic dna hybridization of coding pts protein, and then the expression of arrestin matter, for example, suppress to transcribe and/or translate.Hybridization can form stable duplex by conventional Nucleotide is complementary, and perhaps, for example, when antisense nucleic acid molecule during in conjunction with the dna double spiral, special interaction takes place for it and double-helical major groove.Antisense molecule can be modified, thereby makes this molecule to combine with acceptor or with the antigen-specific of specific cells surface expression, and for example, antisense nucleic acid molecule combines with polypeptide or antibody, and this antibody combines with cell surface receptor or antigen.Antisense nucleic acid molecule also can use carrier described herein to be delivered to cell.In order to obtain the antisense molecule of enough concentration in the cell, such carrier is preferred, and promptly in this carrier, antisense nucleic acid molecule is placed under the control of protokaryon, virus or eukaryotic promoter.

And in another embodiment, antisense nucleic acid molecule of the present invention is a kind of α-anomer nucleic acid molecule.α-anomer nucleic acid molecule and complementary RNA form special double-stranded crossbred, and two strands of chains move towards parallel to each other in the crossbred, this and common β-unit opposite (Gaultier et al. (1987) Nucleic Acids.Res.15:6625-6641).Antisense nucleic acid molecule also can comprise 2 '-o-methyl ribonucleotides (Inoue et al. (1987) Nucleic Acids.Res.15:6131-3148) or chemical RNA-DNA analogue (Inoue et al. (1987) FEBS Lett.215:327-330).

And in another embodiment, antisense nucleic acid molecule of the present invention is a ribozyme.Ribozyme is a catalytic type RNA molecule, has ribonuclease activity, can cutting single-chain nucleic acid, and mRNA for example, it has and single-chain nucleic acid complementary zone.Therefore, ribozyme (for example, hammerhead ribozyme (being described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used for catalyze cleavage PTS mRNA transcript, thereby suppresses the translation of PTS mRNA.For the PTS coding nucleic acid molecule specific ribozyme is arranged, can design based on the PTS dna nucleotide sequence of announcing (being SEQID NO:5 (RAX01503)) herein.For example, can make up the derivative of tetrahymena L-19 IVS RNA, the nucleotide sequence of its avtive spot is a complementary with the nucleotide sequence of the PTS-that is cut coding mRNA.Referring to, for example, 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 can be used for the catalytic type RNA that the screening of RNA library of molecules has special ribozyme activity.Referring to, for example, Bartel, D.and Szostak, J.W. (1993) Science 261:1411-1418.

In addition, as target, form triple-helix structure by handle and PTS nucleotide sequence regulation domain (for example, PTS promotor and/or enhanser) complementary nucleotide sequence, can suppress the PTS expression of gene, this triple-helix structure can stop PTS gene transcribing in the purpose cell.Generally referring to, Helene, C. (1991) Anticancer Drug Des.6 (6): 569-84; Helene, C.et al. (1992) Ann.N.Y.Acad Sci.660:27-36; And Maher, L.J. (1992) Bioassays 14 (12): 807-15.

B. recombinant expression vector and host cell

Another aspect of the present invention relates to carrier, preferably contains coding pts protein (perhaps its part) expression of nucleic acids carrier.Such as used herein, term " carrier " is meant and can connects other nucleic acid, and to its nucleic acid molecule that transports.One type carrier is " plasmid ", and plasmid is meant the circular double-stranded DNA ring, wherein is connected with extra dna fragmentation.The carrier of another kind of type is a virus vector, and wherein extra dna fragmentation can be connected in the viral genome.Some carrier can carry out self-replicating (for example, have the bacteria carrier of bacterium replication orgin, and additive type Mammals carrier) in the host cell that they are introduced into.Other carrier (for example, non-add type Mammals carrier) will be incorporated in the genome of host cell once introducing host cell, thereby together duplicates with host genome.In addition, some carrier can instruct and be attached thereto the expression of gene that connects.These carriers are called " expression vector " herein.In a word, the expression vector of recombinant DNA technology use often is the plasmid form.In this explanation, " plasmid " and " carrier " can exchange use, because plasmid is the carrier format of normal use.Yet the present invention has a mind to comprise other forms of these expression vectors, virus vector (for example, replication defect type retrovirus, adenovirus and adeno associated virus) for example, and they have identical functions.

Recombinant expression vector of the present invention comprises nucleic acid of the present invention, this nucleic acid exists with the form that is fit to expression of nucleic acid in host cell, this just means that recombinant expression vector contains one or more of adjusting sequences, these sequences are based on as the host cell of expressing and select, and they are by feasible being connected on the nucleotide sequence that will express.In recombinant expression vector, the meaning of " feasible connection " is meant, nucleotide sequence interested is connected (for example, in in-vitro transcription/translation system, perhaps in the host cell that carrier is introduced into) with the adjusting sequence in the mode that allows nucleotide sequence to express.The meaning of term " adjusting sequence " is to comprise promotor, enhanser and other expression control elements (for example, polyadenylation signal).This adjusting sequence exists, for example, and Goeddel; GeneExpression Technology:Methods in Enzymology 185, Academic Press, SanDiego, CA has description in (1990).The adjusting sequence comprises that those instruct the sequence of nucleotide sequence constitutive expression in a lot of type host cells, and those instruct the sequence of nucleotide sequence expression in some host cell.The preferred sequence of regulating is, for example, similarly is cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI ^q-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny-, SPO2-, λ-P _R-or λ P _LSuch promotor, these promotors are preferably used in bacterium.Adjusting sequence in addition is, for example, and the promotor of yeast and fungi, ADC1 for example, MF α, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, the promotor of plant, for example, CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, no or ubiquitin-or phaseoin-promotor.Also can use artificial promotor.Person can recognize for being familiar with the routine techniques for these, i.e. the design of expression vector depends on these factors: be used for the selection of transformed host cells, the expression level of desired protein etc.Expression vector of the present invention can be introduced host cell, thereby produces coded protein of nucleic acid described herein or polypeptide, comprises fused protein or polypeptide (for example, the mutant form of pts protein, pts protein, fused protein etc.).

Can design recombinant expression vector of the present invention, be used for expressing pts protein at protokaryon or eukaryotic cell.For example, the PTS gene can be expressed in following cell, it similarly is the such bacterial cell of Corynebacterium glutamicum, insect cell (use 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 ﹠amp; LL.Lasure, eds., p.396-428:Academic Press:San Diego; And van denHondel, C.A.M.J.J.﹠amp; 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 are (referring to Schmidt, R.and Willmitzer, L. (1998) High efficiencyAgrobacterium tumefaciens-mediated transformation of Arabidopsisthaliana leaf and cotyledon explants " Plant Cell Rep.:583-586), perhaps mammalian cell.Appropriate host cell is at Goeddel, Gene Expression Technology:Methods in Enzymology 185, and Academic Press, San Diego, CA has further argumentation in (1990).In addition, recombinant expression vector can for example use the T7 promotor to regulate sequence and T7 polysaccharase in in-vitro transcription and translation.

Protein expression in the prokaryotic cell prokaryocyte often is to be undertaken by the carrier that contains composing type or inducible promoter, and these promotors instruct the expression of fused protein or non-fused protein.Fusion vector adds the amino acid of certain number on coded protein, normally at the N-terminal of recombinant protein.This fusion vector has 3 typical use: 1) increase the expression of recombinant protein; 2) solvability of increase recombinant protein; With 3) as the aglucon of affinity purification, help the fusion rotein purifying.In fusion expression vector, the protein cleavage site often is to be introduced in the junction of merging part and recombinant protein, make after being purified into fused protein, can recombinant protein with merge part and separate.This kind of enzyme, and their cognate recognition sequence comprise Xa factor, zymoplasm and enteropeptidase.

Typical fusion expression vector comprises pGEX (Pharmacia Biotech Inc; Smith, D.B.and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), they have merged glutathione S-transferring enzyme (GST) with target recombinant protein respectively, maltose E is conjugated protein, perhaps a-protein.In one embodiment, the pts protein encoding sequence is cloned in the pGEX expression vector, produces the carrier of an encoding fusion protein, and this carrier comprises to the C-end from the N-end, GST-zymoplasm cleavage site-X protein matter.Fusion rotein can use glutathione-agarose resin, passes through affinitive layer purification.With the reorganization pts protein that GST separates, can be by obtaining with the zymoplasm splitting fused protein.

The example of suitable intestinal bacteria induction type non-fusion expression carrier comprises, 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 and pET 11d (Studier et al., Gene ExpressionTechnology:Methods in Enzymology 185, Academic 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, and depends on the transcribing of host RNA polysaccharase of hybridization trp-lac promoter, fusion.The target gene of pET 11d carrier is expressed, and depends on the transcribing of T7gn10-lac promoter, fusion of viral rna polymerase (T7 gn1) mediation of coexpression.This varial polymerases is provided by resident lambda particles phage among host strain BL21 (DE3) or the HMS174 (DE3), and this phage contains the T7 gn1 gene under the control of lacUV 5 promoter transcriptions.For the conversion of other kinds bacterioid, can select suitable carriers.For example, known plasmid pIJ101, it is effectively that pIJ364, pIJ702 and pIJ361 transform streptomycete, and plasmid pUB110, pC194, perhaps pBD214 is fit to transform shaft-like bacterial classification.Help that genetic information is changed over to coryneform several plasmid and comprise pHM1519, pBL1, pSA77 or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors, Elsevier:NewYork IBSN 0 444 904018).

A kind of scheme that increases expression of recombinant proteins to greatest extent is, in host cell, express such protein, this protein has the ability (Gottesman of the proteolytic cleavage recombinant protein that can not weaken, S., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).Another kind of scheme is to change the nucleotide sequence that inserts the expression vector amplifying nucleic acid, make each amino acid whose codon all be that the selected bacterium that is used to express is preferential and use, for example Corynebacterium glutamicum (Wada et al. (1992) Nucleic Acids Res.20:2111-2118).This change of nucleotide sequence of the present invention can be undertaken by the standard DNA synthetic technology.

In another embodiment, the pts protein expression vector is a Yeast expression carrier.The example of the carrier that yeast S.cerivisae is used to express comprises, 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), and pYES2 (Invitrogen Corporation, San Diego, CA).Be used for structure and be adapted at other fungies, for example in the filamentous fungus, the carrier of the carrier of use and method comprise that those are specified in the following document: van den Hondel, C.A.M.J.J.﹠amp; 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:Cambridge, and Pouwels et al.; eds. (1985) Cloning Vectors, Elsevier:New York IBSN 0 444 904018).

In addition, pts protein of the present invention can use rhabdovirus expression vector in expressed in insect cells.In the insect cell of cultivating (for example Sf9 cell), the baculovirus vector that is used for marking protein comprises, pAC series (Smith et al. (1983) Mol.Cell Biol.3:2156-2165) and pVL series (Lucklow and Summer (1989) Virology 170:31-39).

In another embodiment, pts protein of the present invention can be expressed in one-celled plants cell (for example algae), perhaps expresses in the vegetable cell of higher plant (for example spermatophyte similarly is a crop plants).The example of plant expression vector comprises that those are specified in the following document: 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, comprise pLGV23, pGHlac+, pBIN19, pAK2004 and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors.Elsevier:New York IBSN 0 444 904018).

Also be in another embodiment, nucleic acid of the present invention uses mammalian expression vector to express in mammalian cell.The example of mammalian expression vector comprises pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.6:187-195).The controlled function of expression vector, when being used in the Mammals at that time, often regulating element by virus provides.For example, normally used promotor is from polyoma, adenovirus 2, cytomegalovirus and simian virus 40.Other are for prokaryotic cell prokaryocyte and all suitable expression system of eukaryotic cell, referring to Sambrook, J., Fritsh, E.F., and Maniatis, T.Molecular Cloning:ALaboratory Manual.2nd ed.Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989 16 Zhanghe, 17 chapters.

In another embodiment, the mammalian expression vector of reorganization can instruct the expression (for example, tissue specificity is regulated element and is used to express nucleic acid) of preferred nucleic acid in the particular cell types.It is known technically that tissue specificity is regulated element.The example of suitable tissue-specific promoter including, but not limited to, (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) the Cell 33:729-740 of special promoter of T-cell receptors (Winoto and Baltimore (1989) EMBO is J.8:729-933) and immunoglobulin (Ig); Queen and Baltimore (1983) Cell 33:741-748), the promotor of neuron-specific (neurofilament promotor for example; Byrne and Ruddle (1989) PANS86:5473-5477), the promotor (Edlund et al. (1985) Science 230:912-916) that pancreas is special, and the special promotor of mammary gland (milk whey promotor for example; U.S.Patent No.4,873,316 and European Application Publication No.264,166).Also comprise and grow the promotor of regulating, for example muroid hox promotor (Kessel and Gruss (1990) Science249:374-379) and α-fetoprotein promotor (Campes and Tilghman (1989) Genes Dev.3:537-546).

The present invention provides the recombinant expression vector that contains dna molecular of the present invention in addition, and this dna molecular is cloned in the expression vector with antisense orientation.That is to say that dna molecular can being connected in the following manner of operability be regulated on the sequence, i.e. the mode of the RNA developed by molecule (by transcribing of dna molecular) of permission and PTS mRNA antisense.Can select those in various cell types, to instruct the adjusting sequence of antisense rna molecule continuous expression, for example viral promotors and/or enhanser perhaps can be selected the adjusting sequence that instructs successive, sense-rna tissue-specific or that cell type is special to express, as regulating sequence.Antisense expression vector can exist with the form of recombinant plasmid, phagemid or attenuated virus, and antisense nucleic acid produces under the control of efficient regulation domain therein, and its activity can be determined by the cell type of introducing carrier.About using the inverted defined gene regulatory gene to express, can be referring to Weintraub, H.et al., Antisense RNA as amolecular tool for genetics analysis, Review-Trends in Genetics, Vol.1 (1) 1986.

Another aspect of the present invention relates to the host cell that is introduced into recombinant expression vector of the present invention.Term " host cell " and " recombinant host cell " can be used alternatingly herein.This term is construed as, and not only refers to selected specific cells, and refers to the offspring of these cells or possible offspring.Because sudden change or environmental influence can make some modification occur in the going down to posterity of success, these progeny cells in fact can not be identical with parent cell, but be also contained within the term scope of herein using.

Host cell can be any protokaryon or eukaryotic cell.For example, pts protein can similarly be in the such bacterial cell of Corynebacterium glutamicum, in the insect cell, in the yeast cell or the middle expression of mammalian cell (for example Chinese rat ovary cell (CHO) or COS cell).Other proper host cell, the person knows for being familiar with the routine techniques.Can in table 3, list as the Corynebacterium glutamicum relationship microorganism of nucleic acid of the present invention and protein molecule host cell.

Carrier DNA can transform or rotaring dyeing technology by conventional, introduces protokaryon or eukaryotic cell.Such as used herein, the meaning of term " conversion " and " transfection " is meant various well known in the art, exogenous nucleic acid (for example, linear DNA or RNA are (for example, linear carrier or do not have the independent gene structure of carrier)) or the nucleic acid that exists with carrier format (for example, plasmid, phage, phagemid, phagemid, transposon or other DNA) change the technology of host cell over to, comprise calcium phosphate or calcium chloride co-precipitation, the transfection of DEAE-dextran mediation, lipofection, perhaps fax hole.The appropriate method of conversion or transfection host cell, can be at Sambrook, et al. (Molecular Cloning:A Laboratory Manual.2nd, ed.., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY, 1989), and find on other laboratory manuals.

Known, for stable transfection mammalian cell, the expression vector and the rotaring dyeing technology that rely on to use have only sub-fraction to be incorporated into foreign DNA in himself genome.In order to identify and screen these intasomies, the gene of coding selection markers (for example, to antibiotic resistance) is together introduced host cell with interested gene usually.Preferred selection markers comprises that those can give the mark of drug resistance, for example G418, Totomycin and methotrexate.The nucleic acid of coding selection markers can be introduced into host cell with pts protein on same carrier, perhaps introduce host cell on independent carrier.Through being introduced into the nucleic acid stability cells transfected, can use drug screening to identify (for example, can survive with the cell that the selection markers gene merges, other cells are then died).

In order to create the homologous recombination microorganism, preparation contains to the carrier of small part PTS gene, and this gene has disappearance, adds or replaces, thereby changes for example functional destruction, PTS gene.Preferably this is a Corynebacterium glutamicum PTS gene, but it also can be the homologue from the relationship bacterium, or even from Mammals, yeast or insect.In a preferred embodiment, design vector makes according to homologous recombination, and endogenous PTS gene is by functional destruction (that is code function protein of not being on the permanent staff; Be also referred to as " knocking out " carrier).In addition, can design vector, make that endogenous PTS gene is undergone mutation or changed according to homologous recombination, but encode functional protein matter (for example, change the upstream regulation zone, thereby change endogenous PTS expression of gene) still.In homologous recombination vector, reformed PTS Gene Partial is connected with unnecessary PTS nucleic acid in its 5 ' and 3 ' end profile, makes homologous recombination can occur between the endogenous PTS gene of external source PTS gene that carrier carries and microorganism.The PTS nucleic acid that unnecessary side connects has enough length, can with the generation homologous recombination of native gene success.Typically, contain in the carrier several thousand bases side chain DNA (5 ' and 3 ' end) (referring to, for example, Thomas, K.R., and Capecchi, M.R. (1987) Cell 51:503 for a description of homologous recombinationvectors).Introduce the carrier of microorganism (for example fax hole) and cell, select those the PTS genes wherein introduced and endogenous PTS gene, what known technology can homologous recombination on the use technology.

In another embodiment, can produce the recombinant microorganism that contains the system of being allowed a choice, this system allows to regulate the introducing expression of gene.For example, the PTS gene that comprises is in carrier under the control of lac operon, makes the PTS gene to express when IPTG exists.This regulation system is known technically.

In another embodiment, the endogenous PTS gene in the host cell destroyed (for example, by known genetic method on homologous recombination or the other technologies) makes the expression of its protein not take place.In another embodiment, endogenous in the host cell or the PTS gene of introducing change through one or more point mutation, disappearance or inversion, but encoding function pts protein still.And in another embodiment, one or more regulation domain of microorganism PTS gene (for example, promotor, repressor or elicitor) be changed (for example) by disappearance, shearing, inversion or point mutation, make that the PTS expression of gene is adjusted.The person can recognize to be familiar with the routine techniques, contains the host cell of a more than described PTS gene and protein modification, uses method of the present invention to produce easily, and these cells are also contained among the present invention.

Host cell of the present invention, for example protokaryon of Pei Yanging or eukaryotic host cell can be used for producing (for example expressing) pts protein.Therefore, the present invention further provides, used host cell of the present invention to produce the method for pts protein.In one embodiment, this method is included in cultivation host cell of the present invention in the suitable medium and (has wherein introduced the recombinant expression vector of coding pts protein, perhaps introduced the gene of the pts protein of encoding wild type or change in its genome), up to producing pts protein.In another embodiment, this method further comprises from substratum or host cell and separates pts protein.

C. isolating pts protein

Another aspect of the present invention relates to isolating pts protein and biologically-active moiety thereof." isolating " or " purifying " albumen, perhaps its biologically-active moiety does not have cellular material basically when using recombinant DNA technology to produce, and does not have precursor or other chemical substances when chemosynthesis basically.Term " is substantially free of cellular material " and comprises such pts protein preparation, and wherein protein is isolated from the cellular component of natural or this proteinic cell of reorganization generation.In one embodiment, term " is substantially free of cellular material " and comprises that preparation contains the pts protein of the non-pts protein of about at least 30% (dry weight) (being also referred to as " contaminating protein matter " herein), preferred containing is less than about 20% non-pts protein, even preferred containing be less than about 10% non-pts protein, most preferred containing is less than about 5% non-pts protein.When pts protein or its biologically-active moiety when reorganization produces, preferably be substantially free of substratum, promptly substratum is less than the about 20% of preparation protein volume, preferably is less than 10%, most preferredly is less than about 5%.Term " is substantially free of precursor or other chemical substances " and comprises such pts protein preparation, and wherein protein is isolated from the precursor that participates in protein synthesis or other chemical substances.In one embodiment, term " is substantially free of precursor or other chemical substances " and comprises that preparation contains the pts protein of about at least 30% (dry weight) precursor or non-PTS chemical substance, preferred containing is less than about 20% precursor or non-PTS chemical substance, even preferred containing be less than about 10% precursor or non-PTS chemical substance, most preferred containing is less than about 5% precursor or non-PTS chemical substance.In a preferred embodiment, isolating protein or its biologically-active moiety do not contain the contaminating protein matter from the same organism that obtains pts protein.This protein is typically by recombinant expressed generation, for example the Corynebacterium glutamicum pts protein in the microorganism recombinant expressed as Corynebacterium glutamicum.

Isolating pts protein of the present invention or its biologically-active moiety, can participate in handle similarly is that the such high-energy carbon-containing molecules of glucose is transported into Corynebacterium glutamicum, perhaps participate in signal conduction in the cell in this microorganism, perhaps have one or more and be listed in activity in the table 1.In a preferred embodiment, protein or its part contain such aminoacid sequence, this sequence and aminoacid sequence of the present invention are (for example, sequence table even number sequence number a: sequence in the sequence) sufficient homology is arranged, make this protein or its biologically-active moiety, have the ability to participate in similarly being that the such high-energy carbon-containing molecules of glucose is transported into Corynebacterium glutamicum, perhaps participate in signal conduction in the cell in this microorganism.Proteinic part preferably is meant biologically-active moiety described herein.In another preferred embodiment, pts protein of the present invention has in sequence table with the even number sequence number: the aminoacid sequence of listing.In another preferred embodiment, pts protein has by nucleotide sequence coded aminoacid sequence, this nucleotide sequence and nucleotide sequence of the present invention (for example, sequence table odd serial numbers a: sequence in the sequence) hybridization, for example hybridize under stringent condition.In another preferred embodiment, pts protein has by so nucleotide sequence coded aminoacid sequence, and this nucleotide sequence and a nucleotide sequence of the present invention or its part have about at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% homology preferably has about at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% homology, preferred have about at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90%, perhaps 91%, 92%, 93%, 94%, and even preferredly have about at least 95%, 96%, 97%, 98%, 99% or higher homology.Scope between above-cited value or consistence value (for example, the consistence of 70-90% or the consistence of 80-95%), that also has a mind to comprises in the present invention.For example, that has a mind to has comprised such consistence value scope, and these scopes are combinations of the above-cited upper limit and/or lower value.The preferred pts protein of the present invention also preferably has at least a PTS activity described herein.For example, the preferred pts protein of a kind of the present invention comprises so nucleotide sequence coded aminoacid sequence, this nucleotide sequence and nucleotide sequence hybridization of the present invention, hybridize under stringent condition for example, and it similarly is that the such high-energy carbon-containing molecules of glucose is transported into Corynebacterium glutamicum that this sequence can participate in handle, perhaps participate in signal conduction in the cell in this microorganism, perhaps have one or more and be listed in activity in the table 1.

In other embodiments, pts protein and aminoacid sequence of the present invention are (for example, sequence table even number sequence number a: sequence in the sequence) sufficient homology is arranged, and has the proteinic functionally active of aminoacid sequence of the present invention, as above I partly describe in detail, its aminoacid sequence is because natural change or sudden change and different.Therefore, in another embodiment, pts protein is such protein, and aminoacid sequence that it has and complete aminoacid sequence of the present invention have about at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60% homology preferably has about at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70% homology, preferred have about at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 88%, 89% or 90%, perhaps 91%, 92%, 93%, 94%, and even preferredly have about at least 95%, 96%, 97%, 98%, 99% or higher homology, and have at least a PTS activity described herein.Scope between above-cited value or consistence value (for example, the consistence of 70-90% or the consistence of 80-95%), that also has a mind to comprises in the present invention.For example, that has a mind to has comprised such consistence value scope, and these scopes are combinations of the above-cited upper limit and/or lower value.In another embodiment, the present invention is relevant with such total length Corynebacterium glutamicum protein, and this protein and aminoacid sequence of the present invention have sufficient homology.

The biologically-active moiety of pts protein comprises such polypeptide, this polypeptide contains the aminoacid sequence from the pts protein aminoacid sequence, for example, sequence table even number sequence number: aminoacid sequence or with the aminoacid sequence of pts protein homologous protein, this part contains than total length pts protein or total length pts protein homologous protein amino acid still less, and shows at least a pts protein activity.Typical biologically-active moiety (peptide, for example, amino acid length is for similarly being 5,10,15,20,30,35,36,37,38,39,40,50,100 or more peptide) comprise that one has at least a pts protein active structures territory or primitive.In addition, the other biological active part, wherein proteinic other parts are deleted, can prepare by recombinant technology, and identify its one or more activity described herein.The biologically-active moiety of preferred pts protein contains structural domain/primitive or its part of one or more biologically active of selecting.

Pts protein is preferably by recombinant DNA technology production.For example, the cloned nucleic acid molecule of coded protein (as mentioned above) in expression vector, expression vector is introduced host cell (as mentioned above) and express pts protein in host cell.According to suitable purification scheme, use standard protein purification technique then, from cell, separate pts protein.Except recombinant expressed, can use standard peptide synthetic technology chemosynthesis pts protein, polypeptide or peptide.In addition, natural pts protein can separate from cell (for example endotheliocyte), for example uses anti--PTS antibody, and this antibody can use pts protein of the present invention or its part to produce by standard technique.

The present invention also provides PTS chimeric protein or fusion rotein.As used herein, PTS " chimeric protein " or " fusion rotein " contain operability and are connected to PTS polypeptide on the non-PTS polypeptide." PTS polypeptide " is meant and contains PTS related amino acid polypeptide of sequence, and " non-pts protein " is meant and contains such protein related amino acid polypeptide of sequence, this protein and pts protein do not have basic homology, for example, from the protein different of identical or different organisms with pts protein.In fused protein, the meaning of term " operability connection " is meant that pts protein and non-pts protein are in-frame fusion each other.Non-PTS polypeptide can be fused to the N-end or the C-end of PTS polypeptide.For example, in one embodiment, fused protein is the DST-PTS fusion rotein, and wherein the PTS sequence is fused to the C-end of GST sequence.This fused protein help to recombinate purifying of pts protein.In another embodiment, fused protein is the pts protein that the allos signal sequence is arranged at its N-end.In some host cell (for example mammalian host cell), can increase the expression and/or the secretion of pts protein by using the allos signal sequence.

Preferably, chimeric protein of the present invention or fusion rotein produce by the standard recombinant dna technology.For example, linked together by in-frame according to the encode dna fragmentation of different peptide sequences of routine techniques, for example, use the end of blunt end or staggered end to connect, use Restriction Enzyme to digest so that suitable end to be provided, it is flat as suitable end to use sticky end to mend, and uses alkaline phosphatase treatment avoiding undesirable connection, and uses enzymatic to connect.In another embodiment, can use routine techniques to comprise that automatic dna synthesizer synthesizes fusion gene.In addition, can use the anchor primer to carry out the pcr amplification of gene fragment, the anchor primer can increase by two complementary overhangs between the consecutive gene fragment, consecutive gene can anneal subsequently and again increase and produce chimeric gene sequence (referring to, for example, Current Protocols in MolecularBiology, eds.Ausubel et al.John Wiley ﹠amp; Sons:1992).In addition, the expression vector that merges part (for example gst polypeptide) of much having encoded is that commerce provides.The PTS-coding nucleic acid can be cloned in this expression vector, make to merge that part is in-frame to be connected on the pts protein.

The homologue of pts protein can produce by sudden change, for example sudden change of the point of discontinuity of pts protein or shearing.As used herein, term " homologue " is meant the variant form of pts protein, and they can be used as active agonist of pts protein or antagonist.The agonist of pts protein can have pts protein biological activity identical or part basically.The antagonist of pts protein can suppress one or more activity of the natural existence form of pts protein, for example, and by combining with the downstream or the upstream member competitiveness of the PTS system that comprises pts protein.Therefore, Corynebacterium glutamicum pts protein of the present invention and homologue thereof, the activity that can regulate one or more of carbohydrate transporting pathway, the activity of perhaps regulating signal transduction path in the cell that pts protein plays a role in this microorganism.

In other embodiments, the homologue of pts protein can for example be sheared mutant by the combinatorial library of screening pts protein mutant, identifies pts protein agonist or antagonistic activity.In one embodiment, the diverse libraries of PTS variant produces by the sudden change of associativity on nucleic acid level, and is encoded by diversity gene library.The diverse libraries of PTS variant can pass through, for example, synthetic oligonucleotide mixture enzymatic is connected in the gene order, make the degeneracy of potential PTS sequence gather as single polypeptide, perhaps wherein contain the set of the bigger fused protein (for example for phage display) of PTS arrangement set, can express.There is the whole bag of tricks can be used for, produces potential PTS homologue library from degeneracy oligonucleotide sequence.Can carry out the chemosynthesis of degeneracy gene order with automatic dna synthesizer, synthetic gene is connected in the suitable expression vector then.The use of gene degeneracy set allows blended that the full sequence of the required potential PTS arrangement set of coding is provided.The method of synthetic degeneracy oligonucleotide be technically known (referring to, for example, 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 segmental library of coding pts protein can be used for producing the segmental diversity of PTS colony, and this colony is used to screen and select the homologue of pts protein.In one embodiment, the segmental library of encoding sequence can produce like this, promptly only produce under the condition of an otch at about per molecule, handle the double-stranded PCR fragment of PTS encoding sequence with nuclease, denatured double stranded dna, renaturation DNA to be forming double-stranded DNA, and this double-stranded DNA can comprise from what difference had that the product of otch forms has justice/antisense right, in the duplex that forms again, remove the strand part, and the fragment library that obtains at last is connected in the expression vector by the processing of S1 nuclease.By this method, the N-end that can obtain encoding, C-is terminal and the expression library of different big or small pts protein intermediate segment.

Many technology of the gene product in the combinatorial library that screening is obtained by point mutation or shearing, and the technology that has the characteristic gene product of selecting in the screening cDNA library all are known technically.These technology all are applicable to the rapid screening of the gene library that is obtained by PTS homologue combinatorial mutagenesis.Screen the most widely used technology of large-scale gene pool, can be used in high throughput analysis, comprise genomic library is cloned in the reproducible expression vector, with the vector library conversion carrier library that obtains, and express combination gene under certain condition, the required active detection separation of carrier of detected product gene that helps to encode under this condition.Return assemblage sudden change (REM), a kind of new technology that increases function mutation body frequency in the library can be analyzed one with screening and be used from evaluation PTS homologue (Arkin and Yourvan (1992) PANS 89:7811-7815; Delgrave et al. (1993) Protein Engineering6 (3): 327-331).

In another embodiment, known method on the use technology can be used to analyze diversity PTS library based on the analysis of cell.

D. application of the present invention and method

Nucleic acid molecule described herein, protein, protein homology thing, fused protein, primer, carrier and host cell can be applied in following one or more methods: identify Corynebacterium glutamicum and relationship microorganism; Draw the Genome Atlas of Corynebacterium glutamicum relationship organism; Identify and locate the sequence interested of Corynebacterium glutamicum; Study on Evolution; Determine the essential zone of function of pts protein; Pts protein is active to be regulated; The PTS pathway activities is regulated; Required compound, for example adjusting of the cells produce of fine chemistry material.

PTS nucleic acid molecule of the present invention has various uses.At first, they can be used to identify whether a kind of organism is Corynebacterium glutamicum or its close relative organism.They also can be used for identifying the existence of mixing microorganisms colony Corynebacterium glutamicum or its relationship organism.The invention provides the nucleotide sequence of many Corynebacterium glutamicum genes; Under stringent condition, use the probe of crossing over the Corynebacterium glutamicum specific gene, survey the genomic dna that from single or mixing microorganisms culture, extracts, can determine whether this organism exists.

Although Corynebacterium glutamicum itself is a non-virulent, it is relevant with pathogenic kind, for example corynebacterium diphtheriae.Corynebacterium diphtheriae is the etiology of diphtheria, and diphtheria is a kind of infection rapid, acute, fever that develops, and it relates to local symptom and system's symptom.During this disease, upper respiratory tract generation local patholoic change, and comprise the damage of epithelial cell gangrenosum acne; Bacterium excretes poison, and toxin spreads to the easily infected tip tissue of health from lesion.These tissues comprise heart, muscle, peripheral nerve, suprarenal gland, kidney, liver and spleen, sexually revise owing to protein synthesis is suppressed cause rotten therein, can cause system's symptom of this disease.Many areas keep high incidence to diphtheria in the world, and these areas comprise the independent state of Africa, Asia, Eastern Europe and USSR (Union of Soviet Socialist Republics).From nineteen ninety, lasting popular in latter two regional diphtheria caused at least 5,000 people's death.

In one embodiment, the present invention with identify the experimenter in corynebacterium diphtheriae exist or active method relevant.This method comprises identifies one or more of nucleic acid of the present invention or aminoacid sequence (for example, being listed in odd number or even number sequence number sequence in the sequence table respectively) among the experimenter, thereby detects the existence or the activity of corynebacterium diphtheriae among the experimenter.Corynebacterium glutamicum and corynebacterium diphtheriae are akin bacteriums, and many nucleic acid in the Corynebacterium glutamicum and protein molecule are the homologues of corynebacterium diphtheriae amplifying nucleic acid and protein molecule, therefore also can be used for detecting experimenter's corynebacterium diphtheriae.

Nucleic acid of the present invention and protein molecule also can be as the marks of genome specific region.This is not only useful when drawing Genome Atlas, and can be used for the research of Corynebacterium glutamicum protein function.For example,, can digest the Corynebacterium glutamicum gene group, with fragment and DNA is conjugated protein hatches in order to identify the conjugated protein bonded with it of specific Corynebacterium glutamicum DNA genome area.Can further survey with the fragment of protein bound, preferably use easy certification mark with nucleic acid molecule of the present invention; These nucleic acid molecule combine with genomic fragment, can locate the position of fragment on Corynebacterium glutamicum gene picture group spectrum, and, when using different enzymes to carry out multi-pass operations, help to determine fast protein bonded nucleotide sequence with it.In addition, nucleic acid molecule of the present invention can have sufficient homology with the relationship kind, makes these nucleic acid molecule can be used as the mark that makes up relationship bacterial genomes collection of illustrative plates, for example brevibacterium.

PTS nucleic acid molecule of the present invention can be used for again evolving and protein structure research.The carbohydrate capturing system that molecule of the present invention participates in is used by various bacterium; By comparing the sequence of nucleic acid molecules of sequence of nucleic acid molecules of the present invention and those similar enzymes of in the other biological body, encoding, can estimate the evolution dependency of organism.Similarly, this estimation conserved sequence region and the non-conserved sequence region of relatively allowing, this can help to determine in the protein for the essential zone of enzyme function.Suchly determine that research is valuable for protein engineering, and can indicate those protein to stand to suddenly change and do not lose function.

The operation of PTS nucleic acid molecule of the present invention can cause having the generation with the pts protein of wild-type pts protein difference in functionality.Can improve these proteinic efficient or activity, can make it perhaps to reduce its efficient or activity to appear in the cell than usually more number.

The invention provides screening and can regulate the method for the active molecule of pts protein, these molecules or by with protein itself or substrate interaction, perhaps combine, perhaps by regulating transcribing or translate and regulating the pts protein activity of PTS nucleic acid molecule of the present invention with the mating partner of pts protein.In the method, express the microorganism of one or more pts proteins, contact, and assess of the effect of every kind of test compounds for pts protein activity or expression level with one or more test compounds.

PTS molecule of the present invention can be modified, and makes output, production and/or the production efficiency of one or more chemical substances be improved.For example, its activity is optimized, can increases glucose uptake amount or glucose and be transported the into speed of cell by modifying the pts protein that participates in glucose uptake.The degraded of glucose and other carbohydrates in the cell provides energy to promote the disadvantageous biochemical reaction of energy, and for example those relate to the biosynthetic reaction of fine chemistry material.Degraded also provides the biosynthesizing necessary intermediate of some fine chemistry material or precursor molecule, for example amino acid, VITAMIN and cofactor.By modifying PTS molecule of the present invention to increase the quantity of high-energy carbon molecule in the cell, thereby can both increase the energy of producing the necessary execution pathways metabolism of one or more fine chemistry materials, can increase the needed endocellular metabolism thing of this production storehouse again.Opposite, the degraded product of some carbohydrate contains a kind of compound, this compound only is used for such pathways metabolism, this approach is vied each other with the approach that is used as the required fine chemistry material of generation because of enzyme, cofactor or intermediate, by reducing the input of these carbohydrates, can bear this approach of adjusting.

In addition, PTS molecule of the present invention can participate in signal transduction path in one or more cells, and these approach can influence the output and/or the production efficiency of one or more fine chemistry materials in the Corynebacterium glutamicum.For example, in case there is the carbohydrate of sufficient amount in the cell, the necessary protein of one or more carbohydrates of input (for example from extracellular medium, HPr, Enzyme I, the perhaps a kind of composition in the Enzyme II complex body) often is translated the back and modifies, thereby they can not be input to sugar in the cell again.An example like this appears in the intestinal bacteria, and in the cell 1, the high level of 6-hexose diphosphate causes the phosphorylation of HPr Serine-46, makes this molecule can not participate in the transhipment of any carbohydrate again.Yet, in this endocellular sugar class level that movement system is closed, be enough for the normal function of keeping cell, this may limit the excessive production of required fine chemistry material.Therefore, it is desirable modifying pts protein of the present invention like this, promptly make them no longer effective to this negative adjusting, thereby allow to reach the higher IC of one or more carbohydrates, and, expand, allow from the organism that contains this sudden change pts protein, to obtain more effective production of one or more fine chemistry materials or higher output.

The above-mentioned tabulation that causes the PTS sudden change scheme that required compound output increases, and do not mean that and only be confined to this; The variation of these sudden change schemes knows better for the person skilled in the art.Through these mechanism, nucleic acid of the present invention and protein molecule can be used to produce Corynebacterium glutamicum or its relationship bacterial strain of expressing sudden change PTS nucleic acid and protein molecule, thereby increase output, production and/or the production efficiency of required compound.This required compound can be any natural product of Corynebacterium glutamicum, this comprises the final product and the natural intermediate that has pathways metabolism of biosynthetic pathway, and is not the molecule that natural existence is still produced by Corynebacterium glutamicum strain of the present invention in the Corynebacterium glutamicum metabolism.

The present invention is further by following Examples set, and these examples should not be interpreted as only being confined to this.Content in all reference of being quoted among the application, patent application, patent, the patent application of delivering, table and the sequence list is all integrated with reference hereby.

Table 1: the gene that the present invention includes

Phosphoenolpyruvic acid: carbohydrate phosphotransferase system

Nucleotide SEQ?ID?NO	Amino acid SEQ?ID?NO	Numbering?	Contig?	NT is initial?	NT stops?	Function?
Nucleotide SEQ?ID?NO	Amino acid SEQ?ID?NO	Numbering?	Contig?	NT is initial?	NT stops?	Function?	1 ? 3 ? 5 ? 7 9 11 13 15 ? 17 19 21 23 ? 25 27 29 31 33	2 ? 4 ? 6 ? 8 10 12 14 16 ? 18 20 22 24 ? 26 28 30 32 34	RXS00315 ? F?RXA00315 ? RXA01503 ? RXN01299 F?RXA01299 F?RXA01883 F?RXA01889 RXA00951 ? RXN01244 F?RXA01244 RXA01300 RXN03002 ? RXC00953 RXC03001 RXN01943 F?RXA02191 F?RXA01943	? ? GR00053 ? GR00424 ? W0068 GR00375 GR00538 GR00540 GR00261 ? W0068 GR00359 GR00375 W0236 ? W0260 ? W0120 GR00642 GR00557	? ? 6537 ? 10392 ? 11954 6 2154 77 564 ? 14141 4837 637 1437 ? 1834 ? 4326 3395 3944	? ? 5452 ? 10640 ? 9891 446 2633 631 172 ? 15844 3329 903 1844 ? 1082 ? 6374 4633 3540	The PTS system, sucrose-specificity IIABC composition (EIIABC-SCR) (sucrose-permease IIABC composition (phosphotransferase II, the ABC composition) (EC 2.7.1.69) PTS system, BETA-glucosides-specificity IIABC composition (EIIABC-BGL) (BETA-glucosides-permease IIABC composition) (phosphotransferase II, the ABC composition) (EC 2.7.1.69) PTS system, BETA-glucosides-specificity IIABC composition (EIIABC-BGL) (BETA-glucosides-permease IIABC composition) (phosphotransferase II, the ABC composition) (EC 2.7.1.69) PTS system, fructose-specificity IIBC composition (EC 2.7.1.69) PTS system, fructose-specificity IIBC composition (EC 2.7.1.69) PTS system, fructose-specificity IIBC composition (EC 2.7.1.69) PTS system, fructose-specificity IIBC composition (EC 2.7.1.69) PTS system, N.F,USP MANNITOL (secret)-specificity IIA composition (EIIA-(C) MTL) (N.F,USP MANNITOL (secret)-permease IIA composition) (phosphotransferase II, the A composition) (EC 2.7.1.69) phosphoenolpyruvic acid-protein phosphatase transferring enzyme (EC 2.7.3.9) phosphoenolpyruvic acid-protein phosphatase transferring enzyme (EC 2.7.3.9) phosphate carrier albumen HPR PTS system, N.F,USP MANNITOL (secret)-specificity IIA composition (EIIA-(C) MTL) (N.F,USP MANNITOL (secret)-permease IIA composition) (phosphotransferase II, the A composition) the transmembrane protein PTS system in the transmembrane protein PTS system in (EC 2.7.1.69) PTS system, glucose-specificity IIABC composition (EC 2.7.1.69) phosphoenolpyruvic acid carbohydrate phosphotransferase crr gene; Phosphotransferase system glucose-specific enzymes III

Table 2:GENBANK genes identified

GenBank ^TMSearching number	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	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 dehydra(ta)se	Moeckel，B.et?al.“Production?of?L-isoleucine?by?means?of?recombinant micro-organisms?with?deregulated?threonine?dehydratase，”Patent：WO 9519442-A?5?07/20/95	A09073	ppg	Phosphoric acid enol pyruvic acid carboxylase
A45579， A45581， A45583， A45585 A45587		Threonine dehydra(ta)se		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	The D-glutamate racemase
AB023377	tkt	Transketolase		AB020624	murI	The D-glutamate racemase
AB023377	tkt	Transketolase		AB024708	gltB；gltD	Big and the small subunit of glutamine 2-oxoglutaric acid transaminase
AB025424	acn	Aconitase		AB024708	gltB；gltD
AB025424	acn	Aconitase		AB027714	rep	Replication protein
AB027715	rep；aad	Replication protein; Aminoglycoside adeninyl transferring enzyme		AB027714	rep	Replication protein
AB027715	rep；aad			AF005242	argC	N-acetyl aspartic acid-5-semialdehyde desaturase
AF005635	glnA	Glutamine synthetase		AF005242	argC	N-acetyl aspartic acid-5-semialdehyde desaturase
AF005635	glnA	Glutamine synthetase		AF030405	hisF	Cyclase
AF030520	argG	Argininosuccinate synthetase		AF030405	hisF	Cyclase
AF030520	argG	Argininosuccinate synthetase		AF031518	argF	Ornithine transcarbamylase
AF036932	aroD	The 3-dehydroquinate dehydratase		AF031518	argF	Ornithine transcarbamylase

GenBank ^TM

The gene title

Gene function

Reference

Searching number
Searching number				AF038548	pyc	Pyruvate carboxylase
AF038651	dciAE；apt；rel	Two peptide-binding proteins; Adenine phosphoribosyl transferase; The GTP pyrophosphokinase	Wehmeier，L.et?al.“The?role?of?the?Corynebacterium?glutamicum?rel?gene?in (p)ppGpp?metabolism，”Microbiology，144：1853-1862(1998)	AF038548	pyc	Pyruvate carboxylase
AF038651	dciAE；apt；rel			AF041436	argR	The arginine repressor
AF045998	impA	The inositol monophosphate Starch phosphorylase		AF041436	argR	The arginine repressor
AF045998	impA	The inositol monophosphate Starch phosphorylase		AF048764	argH	The argininosuccinic acid lyase
AF049897	argC；argJ；argB； argD；argF；argR； argG；argH	N-acetyl glutamy phosphoric acid reduction enzyme; Ornithine acetyltransferase; The N-acetylglutamat kinases; Acetylornithice transminase; Ornithine transcarbamylase; The arginine repressor; The argininosuccinic acid synthase; The argininosuccinic acid lyase		AF048764	argH	The argininosuccinic acid lyase
AF049897	argC；argJ；argB； argD；argF；argR； argG；argH			AF050109	inhA	Alkene acyl-acyl carrier protein reductase enzyme
AF050166	hisG	ATP phosphoribosyltransferase		AF050109	inhA	Alkene acyl-acyl carrier protein reductase enzyme
AF050166	hisG	ATP phosphoribosyltransferase		AF051846	hisA	Phosphoribosyl formimino-5-amino-ribose 1-phosphate-4-imidazoles carboxamide 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	The dehydroquinic acid synthetic enzyme
AF060558	hisH	Glutamine acid amides transferring enzyme		AF053071	aroB	The dehydroquinic acid synthetic enzyme
AF060558	hisH	Glutamine acid amides transferring enzyme		AF086704	hisE	Phosphoribosyl-ATP-pyrophosphohydrolase
AF114233	aroA	5-enol pyruvic acid shikimic acid 3-phosphate synthase		AF086704	hisE	Phosphoribosyl-ATP-pyrophosphohydrolase
AF114233	aroA	5-enol pyruvic acid shikimic acid 3-phosphate synthase		AF116184	panD	L-aspartic acid-alpha-decarboxylase 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	The gene title	Gene function	Reference
?GenBank ^TMSearching number	The gene title	Gene function	Reference	?AF124518	aroD；aroE	3-dehydroquinase; Shikimate dehydrogenase
?AF124600	aroC；aroK；aroB；	Chorismate synthase; Shikimate kinase; The 3-dehydroquinic acid		?AF124518	aroD；aroE	3-dehydroquinase; Shikimate dehydrogenase

	pepQ	Synthase; The kytoplasm peptase of inferring
	pepQ	Synthase; The kytoplasm peptase of inferring		AF145897	inhA
AF145898	inhA			AF145897	inhA
AF145898	inhA			AJ001436	ectP	Ectoine, glycinebetaine, proline(Pro) transhipment	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	Tetrahydrochysene 2, the dipicolimic acid 2 succinyl enzyme is (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(Pro) transhipment
AJ004934	dapD			AJ007732	ppc；secG；amt；ocd； soxA	Phosphoenolpyruvic acid-carboxylase; High-affinity ammonia absorbs albumen; Ornithine-ring decarboxylase of inferring; Sarcosine oxidase
AJ010319	ftsY，glnB，glnD；srp； amtP	Participate in cell fission; 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	E.C. 2.3.1.28
AJ224946	mqo	L MALIC ACID: quinone oxidoreductase	Molenaar，D.et?al.“Biochemical?and?genetic?characterization?of?the membrane-associated?malate?dehydrogenase(acceptor)from?Corynebacterium glutamicum，”Eur.J.Biochem.，254(2)：395-403(1998)	AJ132968	cat	E.C. 2.3.1.28
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	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	D84102	odhA	The 2-oxoglutaric acid desaturase	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?110/12/87	D84102	odhA	The 2-oxoglutaric acid desaturase

E01359		Homoserine kinase gene start codon upstream	Katsumata，R.et?al.“Production?of?L-thereonine?and?L-isoleucine，”Patent：JP 1987232392-A?210/12/87
E01359		Homoserine kinase gene start codon upstream		E01375	Tryptophan operon
E01376	trpL；trpE	Leading peptide; The o-amino benzoyl acid 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?110/24/87	E01375	Tryptophan operon
E01376	trpL；trpE	Leading peptide; The o-amino benzoyl acid synthase		E01377	Tryptophan operon promotor 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?110/24/87
E03937		Vitamin H-synthase	Hatakeyama，K.et?al.“DNA?fragment?containing?gene?capable?of?coding biotin?synthetase?and?its?utilization，”Patent：JP?1992278088-A?110/02/92	E01377	Tryptophan operon promotor and operator region
E03937		Vitamin H-synthase		E04040	The 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-A1 11/18/92	E04040	The diaminopelargonic acid transaminase
E04041		Dethiobiotin synthetase		E04307	The Flavum L-Aspartase	Kurusu，Y.et?al.“Gene?DNA?coding?aspartase?and?utilization?thereof，”Patent： JP?1993030977-A?102/09/93
E04376		Isocitrate lyase	Katsumata，R.et?al.“Gene?manifestation?controlling?DNA，”Patent：JP 1993056782-A?303/09/93	E04307	The Flavum L-Aspartase
E04376		Isocitrate lyase		E04377	Isocitrate lyase N-terminal fragment	Katsumata，R.et?al.“Gene?manifestation?controlling?DNA，”Patent：JP 1993056782-A?303/09/93
E04484		Prephenate dehydratase	Sotouchi，N.et?al.“Production?of?L-phenylalanine?by?fermentation，”Patent：JP 1993076352-A?203/30/93	E04377	Isocitrate lyase N-terminal fragment
E04484		Prephenate dehydratase		E05108	E.C. 2.7.2.4.	Fugono，N.et?al.“Gene?DNA?coding?Aspartokinase?and?its?use，”Patent：JP 1993184366-A?107/27/93
E05112		Dihydro-dipichorinate synthetic enzyme	Hatakeyama，K.et?al.“Gene?DNA?coding?dihydrodipicolinic?acid?synthetase and?its?use，”Patent：JP?1993184371-A?107/27/93	E05108	E.C. 2.7.2.4.

GenBank ^TMSearching number	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	E05776	Diaminopimelate dehydrogenase	Kobayashi，M.et?al.“Gene?DNA?coding?Diaminopimelic?acid?dehydrogenase and?its?use，”Patent：JP?1993284970-A?111/02/93
E05779		Threonine synthase	Kohama，K.et?al.“Gene?DNA?coding?threonine?synthase?and?its?use，”Patent： JP?1993284972-A?111/02/93	E05776	Diaminopimelate dehydrogenase

E06110		Prephenate dehydratase	Kikuchi，T.et?al.“Production?of?L-phenylalanine?by?fermentation?method，” Patent：JP?1993344881-A?112/27/93
E06110		Prephenate dehydratase		E06111	The sudden change prephenate dehydratase	Kikuchi，T.et?al.“Production?of?L-phenylalanine?by?fermentation?method，” Patent：JP?1993344881-A?112/27/93
E06146		Acetohydroxy acid synthetase	Inui，M.et?al.“Gene?capable?of?coding?Acetohydroxy?acid?synthetase?and?its use，”Patent：JP?1993344893-A?112/27/93	E06111	The sudden change prephenate dehydratase
E06146		Acetohydroxy acid synthetase		E06825	E.C. 2.7.2.4.	Sugimoto，M.et?al.“Mutant?aspartokinase?gene，”patent：JP?1994062866-A1 03/08/94
E06826		Sudden change E.C. 2.7.2.4. alpha subunit	Sugimoto，M.et?al.“Mutant?aspartokinase?gene，”patent：JP?1994062866-A1 03/08/94	E06825	E.C. 2.7.2.4.
E06826		Sudden change E.C. 2.7.2.4. alpha subunit		E06827	Sudden change E.C. 2.7.2.4. alpha subunit	Sugimnoto，M.et?al.“Mutant?aspartokinase?gene，”patent：JP?1994062866-A1 03/08/94
E07701	secY		Honno，N.et?al.“Gene?DNA?participating?in?integration?of?membraneous protein?to?membrane，”Patent：JP?1994169780-A?106/21/94	E06827	Sudden change E.C. 2.7.2.4. alpha subunit
E07701	secY			E08177	E.C. 2.7.2.4.	Sato，Y.et?al.“Genetic?DNA?capable?of?coding?Aspartokinase?released?from feedback?inhibition?and?its?utilization，”Patent：JP?1994261766-A?109/20/94
E08178， E08179， E08180， E08181， E08182		The E.C. 2.7.2.4. of feedback inhibition-release	Sato，Y.et?al.“Genetic?DNA?capable?of?coding?Aspartokinase?released?from feedback?inhibition?and?its?utilization，”Patent：JP?1994261766-A?109/20/94	E08177	E.C. 2.7.2.4.
E08178， E08179， E08180， E08181， E08182		The E.C. 2.7.2.4. of feedback inhibition-release		E08232	Acetohydroxy acid isomeroreductase	Inui，M.et?al.“Gene?DNA?coding?acetohydroxy?acid?isomeroreductase，” Patent：JP?1994277067-A?110/04/94
E08234	secE		Asai，Y.et?al.“Gene?DNA?coding?for?translocation?machinery?of?protein，” Patent：JP?1994277073-A?110/04/94	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?102/03/95
E08646		The vitamin H synthetic enzyme	Hatakeyama，K.et?al.“DNA?fragment?having?promoter?function?in coryneform?bacterium，”Patent：JP?1995031476-A?102/03/95	E08643	FT transaminase and dethiobiotin synthetase promoter region

GenBank ^TMSearching number

The gene title

Gene function

Reference

E08649		L-Aspartase	Kohama，K.et?al“DNA?fragment?having?promoter?function?in?coryneform bacterium，”Patent：JP?1995031478-A?102/03/95
E08649		L-Aspartase		E08900		Dihydro 2, the dipicolimic acid 2 reductase enzyme	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?there?of，”Patent：JP?1995075579-A?103/20/95	E08900		Dihydro 2, the dipicolimic acid 2 reductase enzyme
E08901		Diaminapimelate decarboxylase		E12594		Serine hydroxymethylase	Hatakeyama，K.et?al.“Production?of?L-trypophan，”Patent：JP?1997028391-A 102/04/97
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, the dipicolimic acid 2 synthetic enzyme	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, the dipicolimic acid 2 synthetic enzyme		E12770		E.C. 2.7.2.4.	Moriya，M.et?al.“Amplification?of?gene?using?artificial?transposon，”Patent： JP?1997070291-A?03/18/97
E12773		Dihydro 2, the dipicolimic acid 2 reductase enzyme	Moriya，M.et?al.“Amplification?of?gene?using?artificial?transposon，”Patent： JP?1997070291-A?03/18/97	E12770		E.C. 2.7.2.4.
E12773		Dihydro 2, the dipicolimic acid 2 reductase enzyme		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?109/02/97
L01508	IlvA	Threonine dehydra(ta)se	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 dehydra(ta)se		L07603	EC?4.2.1.15	3-deoxidation-D-pectinose 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	The big subunit of acetohydroxy acid synthase; The 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	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	L18874	PtsM	The phosphoenolpyruvic acid 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	Isocitrate lyase
L35906	dtxr	The 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	Isocitrate lyase
L35906	dtxr	The 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.“Phylogenetic?analysis?of?the?coryneform?bacteria?by?56 rRNA?sequences，”J.Bacteriol.，169：1801-1806(1987)	M13774		Prephenate dehydratase
M16175	5S?rRNA			M16663	trpE	The o-amino benzoyl acid synthase, 5 ' end	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 ' end	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	The o-amino benzoyl acid synthase, 5 ' end
M16664	trpA	Tryptophan synthetase, 3 ' end		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 insertion sequence	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	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	M85107，		23S rRNA gene insertion sequence	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；bmQ；yhbw	Beta C-S lyase; Branched-chain amino acid absorbs carrier; The albumen yhbw that infers	Rossol，l.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.“Isoleucine?uptake?in Corynebacterium?glutamicum?ATCC?13032is?directed?by?the?bmQ?gene product，”Arch.Microbiol.，169(4)：303-312(1998)
S59299	trp	Leading gene (promotor)	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；bmQ；yhbw
S59299	trp	Leading gene (promotor)		U11545	trpD	Anthranilate phosphoribosyl transferase	O’Gara，J.P.and?Dunican，L.K.(1994)Complete?nucleotide?sequence?of?the Corynebacterium?glutamicum?ATCC?21850?tpD?gene.”Thesis，Microbiology Department，University?College?Galway，Ireland.
U13922	cglIM；cglIR；clgIIR	The II type 5-cytosine(Cyt) methyltransgerase of inferring; The II type restriction enzyme of inferring; I type or the III type restriction enzyme inferred	Schafer，A.et?al.“Cloning?and?characterization?of?a?DNA?region?encoding?a stress-sensitive?restriction?system?from?Corynebacterium?glutamicum?ATCC 13032and?analysis?of?its?role?in?intergeneric?conjugation?with?Escherichia coli，”J.Bacteriol.，176(23)：7309-7319(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(Pro): NADP+5-oxydo-reductase	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 glutamy kinases; Be similar to the special 2-hydroxy acid dehydrogenase of D-isomer	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(Pro): NADP+5-oxydo-reductase

GenBank ^TMSearching number	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	U31281	bioB	The vitamin H synthase	Serebriiskii，I.G.，“Two?new?members?of?the?bio?B?superfamily：Cloning，

			sequencing?and?expression?of?bio?B?genes?ofMethylobacillus?flagellatum?and Corynebacterium?glutamicum，”Gene，175：15-22(1996)
				U35023	?thtR；accBC	Thiosulfate transsulfurase; The acyl-CoA carboxylase	Jager，W.et?al.“A?Corynebacrerium?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?confering?multidrug resistance?in?the?heterologous?host?Escherichia?coli，”J.Bacteriol.， 179(7)：2449-2451(1997)	U35023	?thtR；accBC	Thiosulfate transsulfurase; The acyl-CoA carboxylase
U43535	cmr	Multi-drug resistance albumen		U43536	clpB	Heat shock ATP-is conjugated protein
U53587	aphA-3	3 ' 5 "-aminoglycoside phosphotransferase		U43536	clpB	Heat shock ATP-is conjugated protein
U53587	aphA-3	3 ' 5 "-aminoglycoside phosphotransferase		U89648		Participate in the biosynthetic Corynebacterium glutamicum of Histidine and do 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?ofCorynebacterium glutamicum?and?possible?mechanisms?for?modulation?of?its?expression，”Mol. Gen.Genet，212(1)：112-119(1988)
X14234	EC?4.1.1.31	Keto acid carboxylase in the phosphoric acid enol	Eikmanns，B.J.et?al.“The?Phosphoenolpyruvate?carboxylase?gene?of Corynebacterium?glutamicum：Molecular?cloning，nueleotide?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	Keto acid carboxylase in the phosphoric acid enol		X17313	fda	Fructose-bisphosphate aldolase	Von?der?Osten，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.Microbiol.，
X53993	dapA	L-2,3-dihydro 2, dipicolimic acid 2 synthetic enzyme (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	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	X54223		The 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?Corynebacteriumglutamicum?lysA?gene，”Mol.Microbiol.，4(11)：1819- 1830(1990)
X55994	trpL；trPE	The leading peptide of inferring; O-amino benzoyl acid synthase composition 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	The 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	The attB-related locus	Attachment site		X57226	lysC-alpha；lysC-beta； asd	E.C. 2.7.2.4.-alpha subunit; E.C. 2.7.2.4.-beta subunit; Aspartic acid beta semialdehyde desaturase	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.Bacteriol.，174(19)：6076-6086 (1992)	X57226	lysC-alpha；lysC-beta； asd
X59403	gap；pgk；tpi			X59404	gdh	Glutamate 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-Methionin 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	Glutamate dehydrogenase

GenBank ^TMSearching number	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	X66078	cop1	Psl albumen	Joliff，G.et?al.“Cloning?and?nucleotide?sequence?of?the?csp1?gene?encoding

			PS1，one?of?the?two?major?secreted?proteins?of?Corynebacterium?glutamicum： The?deduced?N-terminal?region?of?PS1?is?similar?to?the?Mycobacterium?antigen 85complex，”Mol.Microbiol.，6(16)：2349-2362(1992)
				X66112 ? ?	glt ? ?	Oxalacetic transacetase	Eikmanns，B.J.et?al.“Cloning?sequence，expression?and?transcriptional analysis?of?the?Corynebacterium?glutamicum?gltA?gene?encoding?citrate synthase，”Microbiol.，140：1817-1828(1994)
X67737	dapB	Dihydro 2, the dipicolimic acid 2 reductase enzyme		X66112 ? ?	glt ? ?	Oxalacetic transacetase
X67737	dapB	Dihydro 2, the dipicolimic acid 2 reductase enzyme		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 ? ?		The relevant factor of inserting of IS3	Bonamy，C.et?al.“Identification?of?IS?1206，a?Corynebacterium?glutamicum IS3-related?insertion?sequence?and?phylogenetic?ahalysis，”Mol.Microbiol.， 14(3)：571-581(1994)	X69103 ? ?	csp2 ? ?	S-layer proteins PS2
X69104 ? ?		The relevant factor of inserting of IS3		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 enzyme (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 enzyme (NADP+)		X72855	GDHA	Glutamate dehydrogenase (NADP+)
X75083， X70584 ?	mtrA ? ?	The 5-methyl tryptophan resistance	Heery，D.M.et?al.“A?sequence?faom?a?tryptophan-hyperproducing?strain?of Corynebacterium?glutamicum?encoding?resistance?to?5-methyltryptophan，” Biochem.Biophys.Res.Commun.，201(3)：1255-1262(1994)	X72855	GDHA	Glutamate dehydrogenase (NADP+)
X75083， X70584 ?	mtrA ? ?	The 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 ? ?	The part isocitrate lyase;	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 ? ?	The part isocitrate lyase;		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?Leeuwenhoeek，64：285-305(1993)
GenBank ^TMSearching number	The gene title	Gene function	Reference	X76875 ? ?		ATP enzyme beta-subunit
GenBank ^TMSearching number	The gene title	Gene function	Reference	X77034	tuf	EF-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	The 16S ribosome-RNA(rRNA)	Rainey，F.A.et?al.“Phylogenetic?analysis?of?the?genera?Rhodococcus?and Norcardia?and?evidence?for?the?evolutionary?origin?of?the?genus?Norcardia from?within?the?radiation?of?Rhodococcus?species，”Microbiol.，141：523-528 (1995)
X81191	gluA；gluB；gluC； gluD	The L-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	The 16S ribosome-RNA(rRNA)
X81191	gluA；gluB；gluC； gluD	The L-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	The 16S ribosome-RNA(rRNA)	Ruimy，R.et?al.“Phylogeny?of?the?genus?Corynebacterium?deduced?from analyses?of?small-subunit?ribosomal?DNA?sequences，”Int.J.Syst.Bacteriol.， 45(4)：740-746(1995)	X81379	dapE	Succinyldiaminopimelate desuccinylase
X82061	16S?rDNA	The 16S ribosome-RNA(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-glutamy phosphoric acid reduction enzyme	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-glutamy phosphoric acid reduction enzyme		X84257	16S?rDNA	The 16S ribosome-RNA(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	The aromatic amino acid permease;	Wehrmann，A.et?al.“Functional?analysis?of?sequences?adjacent?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	The 16S ribosome-RNA(rRNA)

GenBank ^TMSearching number

The gene title

Gene function

Reference

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; E.C. 2.7.2.1	Reinscheid，D.J.et?al.“Cloning，sequence?analysis，expression?and?inactivation of?the?Corynebacterium?glutamicum?pta-ack?operon?encoding phosphotransacetylase?and?acetate?kinase，”Microbiology，l45：503-513(1999)
X89850	attB	Attachment site	Le?Marrec，C.et?al.“Genetic?characterization?of?site-specific?integration functions?ofphi?AAU2?infecting“Arthrobacter?aureus?C70，”J.Bacteriol.， 178(7)：1996-2004(1996)	X89084	pta；ackA	Phosphate acetyltransferase; E.C. 2.7.2.1
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	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	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， molccular?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	Patek，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	The 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	The 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	The ammonia movement system
X93514	betP	The glycinebetaine movement system		X95649	orf4		Patek，M.et?al.“Identification?and?transcriptional?analysis?of?the?dapB-ORF2- dapA-ORF4operon?of?Corynebacterium?glutamicum，encoding?two?enzymes involved?in?L-lysine?synthesis，”Biotechnol.Lett，19：1113-1117(l997)
X96471	lysE；lysG	Methionin output albumen; Albumen is regulated in Methionin output	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	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	X96580	panB；panC；xylB	3-methyl-2-oxy butyrate hydroxymethyl transferases; Pantothenic acid-beta-L-Ala ligase enzyme; 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		Insertion sequence IS1207 and transposase
X96962		Insertion sequence IS1207 and transposase		X99289		Elongation factor 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 factor P
Y00140	thrB	Homoserine kinase		Y00151	ddh	Meso-diaminopimelic acid D-desaturase (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-desaturase (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-acetylmurami-L-Ala ligase enzyme; Divide initial albumen or cell fission albumen; Cell fission albumen	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(Pro) movement system	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(Pro) movement system
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		Phage 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	The gene title	Gene function	Reference
GenBank ^TMSearching number	The gene title	Gene function	Reference	Y12537	proP	Proline(Pro)/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)
			betaine?carrier，EctP，”J.Bacteriol.，180(22)：6005-6012(1998)	Y13221	glnA	Glutamine synthetase 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 synthetase I
Y16642	lpd	Dihydrolipoamide dehydrogenase		Y18059		Rod bacillus phage 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?Brevibacteriumn?lactofermentum： Regulation?of?argS-lysA?cluster?expression?by?arginine，”J. Bacteriol.，175(22)：7356-7362(1993)	Y18059		Rod bacillus phage 304L attachment site
Z21501	argS；lysA			Z21502	dapA；dapB	Dihydro 2, the dipicolimic acid 2 synthase; Dihydro 2, the dipicolimic acid 2 reductase enzyme	Pisabarro，A.et?al.“A?cluster?of?three?genes(dapA，orf2，and?dapB)of Brevibacterium?lactofermentum?encodes?dihydrodipicoIinate?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	The 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	The 16S ribosomal RNA gene
Z49822	sigA	The SigA sigma factor		Z49823	galE；dtxR	UDP-semi-lactosi 4-epimerase catalytic activity; Diphtheria toxin is regulated albumen	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)

¹This gene order is open in listed reference.But obviously openly sequence is long for the sequence that the present invention obtains.Inferring disclosed ATG code of sequence mistake, only is a fragment in actual coding district therefore.

Table 3: can be used for implementing excellent bacillus of the present invention and tyrothricin bacterial strain

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

Tyrothricin	Brevibacterium	21086
Tyrothricin	Brevibacterium	21086				Tyrothricin	Brevibacterium	31269
Tyrothricin	Extension brevibacterium	9174				Tyrothricin	Brevibacterium	31269
Tyrothricin	Extension brevibacterium	9174				Tyrothricin	Extension brevibacterium	19391
Tyrothricin	Extension brevibacterium	8377				Tyrothricin	Extension brevibacterium	19391
Tyrothricin	Extension brevibacterium	8377				Tyrothricin	Separate the paraffin tyrothricin			?11160
Tyrothricin	Kind			717.73		Tyrothricin	Separate the paraffin tyrothricin			?11160
Tyrothricin	Kind			717.73		Tyrothricin	Kind				717.73
Tyrothricin	Kind	14604				Tyrothricin	Kind				717.73
Tyrothricin	Kind	14604				Tyrothricin	Kind	21860
Tyrothricin	Kind	21864				Tyrothricin	Kind	21860
Tyrothricin	Kind	21864				Tyrothricin	Kind	21865
Tyrothricin	Kind	21866				Tyrothricin	Kind	21865
Tyrothricin	Kind	21866				Tyrothricin	Kind	19240
The rod bacillus	Corynebacterium acctoacidophlum	21476				Tyrothricin	Kind	19240
The rod bacillus	Corynebacterium acctoacidophlum	21476				The rod bacillus	Corynebacterium acctoacidophlum	13870
The rod bacillus	Vinegar paddy rod bacillus		B11473			The rod bacillus	Corynebacterium acctoacidophlum	13870
The rod bacillus	Vinegar paddy rod bacillus		B11473			The rod bacillus	Vinegar paddy rod bacillus		B11475
The rod bacillus	Vinegar paddy rod bacillus	15806				The rod bacillus	Vinegar paddy rod bacillus		B11475
The rod bacillus	Vinegar paddy rod bacillus	15806				The rod bacillus	Vinegar paddy rod bacillus	21491
The rod bacillus	Vinegar paddy rod bacillus	31270				The rod bacillus	Vinegar paddy rod bacillus	21491
The rod bacillus	Vinegar paddy rod bacillus	31270				The rod bacillus	Have a liking for acetyl rod bacillus		B3671
The rod bacillus	Produce ammonia rod bacillus	6872			2399	The rod bacillus	Have a liking for acetyl rod bacillus		B3671
The rod bacillus	Produce ammonia rod bacillus	6872			2399	The rod bacillus	Produce ammonia rod bacillus	15511
The rod bacillus	fujiokense	21496				The rod bacillus	Produce ammonia rod bacillus	15511
The rod bacillus	fujiokense	21496				The rod bacillus	Corynebacterium glutamicum	14067
The rod bacillus	Corynebacterium glutamicum	39137				The rod bacillus	Corynebacterium glutamicum	14067
The rod bacillus	Corynebacterium glutamicum	39137				The rod bacillus	Corynebacterium glutamicum	21254
The rod bacillus	Corynebacterium glutamicum	21255				The rod bacillus	Corynebacterium glutamicum	21254
The rod bacillus	Corynebacterium glutamicum	21255				The rod bacillus	Corynebacterium glutamicum	31830
The rod bacillus	Corynebacterium glutamicum	13032				The rod bacillus	Corynebacterium glutamicum	31830
The rod bacillus	Corynebacterium glutamicum	13032				The rod bacillus	Corynebacterium glutamicum	14305
The rod bacillus	Corynebacterium glutamicum	15455				The rod bacillus	Corynebacterium glutamicum	14305
The rod bacillus	Corynebacterium glutamicum	15455				The rod bacillus	Corynebacterium glutamicum	13058
The rod bacillus	Corynebacterium glutamicum	13059				The rod bacillus	Corynebacterium glutamicum	13058
The rod bacillus	Corynebacterium glutamicum	13059				The rod bacillus	Corynebacterium glutamicum	13060
The rod bacillus	Corynebacterium glutamicum	21492				The rod bacillus	Corynebacterium glutamicum	13060
The rod bacillus	Corynebacterium glutamicum	21492				The rod bacillus	Corynebacterium glutamicum	21513
The rod bacillus	Corynebacterium glutamicum	21526				The rod bacillus	Corynebacterium glutamicum	21513
The rod bacillus	Corynebacterium glutamicum	21526				The rod bacillus	Corynebacterium glutamicum	21543
The rod bacillus	Corynebacterium glutamicum	13287				The rod bacillus	Corynebacterium glutamicum	21543
The rod bacillus	Corynebacterium glutamicum	13287				The rod bacillus	Corynebacterium glutamicum	21851
The rod bacillus	Corynebacterium glutamicum	21253				The rod bacillus	Corynebacterium glutamicum	21851
The rod bacillus	Corynebacterium glutamicum	21253				The rod bacillus	Corynebacterium glutamicum	21514
The rod bacillus	Corynebacterium glutamicum	21516				The rod bacillus	Corynebacterium glutamicum	21514
The rod bacillus	Corynebacterium glutamicum	21516				The rod bacillus	Corynebacterium glutamicum	21299
The rod bacillus	Corynebacterium glutamicum	21300				The rod bacillus	Corynebacterium glutamicum	21299
The rod bacillus	Corynebacterium glutamicum	21300				The rod bacillus	Corynebacterium glutamicum	39684
The rod bacillus	Corynebacterium glutamicum	21488				The rod bacillus	Corynebacterium glutamicum	39684
The rod bacillus	Corynebacterium glutamicum	21488				The rod bacillus	Corynebacterium glutamicum	21649

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

The rod bacillus	Kind		?P4446
The rod bacillus	Kind		?P4446		The rod bacillus	Kind	31088
The rod bacillus	Kind	31089			The rod bacillus	Kind	31088
The rod bacillus	Kind	31089			The rod bacillus	Kind	31090
The rod bacillus	Kind	31090			The rod bacillus	Kind	31090
The rod bacillus	Kind	31090			The rod bacillus	Kind	31090
The rod bacillus	Kind	15954		20145	The rod bacillus	Kind	31090
The rod bacillus	Kind	15954		20145	The rod bacillus	Kind	21857
The rod bacillus	Kind	21862			The rod bacillus	Kind	21857
The rod bacillus	Kind	21862			The rod bacillus	Kind	21863

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

FERM: fermentation research institute, Chiba, Japan

NRRL: agricultural research institute preservation center, northern regional research laboratory, Peoria, IL, USA

CECT: Spain typical case's culture collection center, Valencia, Spain

NCIMB: state-run industry and marine microorganism preservation center., Aberdeen, UK

CBS: fungi strain preservation center, Baarn, NL

NCTC: state-run typical culture collection center, London, UK

DSMZ: Germany microbial preservation center, Braunschweig, Germany

Can be referring to 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 comparative result

ID#? ? ?	The Genbank hit results? (NT)?	Length? ? ?	Searching number? ? ?	Genbank hit results title? ? ?	The Genbank hit results The source?	％ Homology (GAP)	Warehouse-in day? ? ?
ID#? ? ?	The Genbank hit results? (NT)?	Length? ? ?	Searching number? ? ?	Genbank hit results title? ? ?	The Genbank hit results The source?	％ Homology (GAP)	Warehouse-in day? ? ?	rxa00315 ? ? ? ? rxa01503 ? ? ? ? ? rxa01299 ? ? ? ? ? rxa00951 ? ? ? ? ? rxa01244 ? ? ? ? ? rxa01300 ? ? rxa00953 ? ? ? ? ?	1527GB_BA1：AB007125 ? GB_IN1：CELC47D2 GB_HTG2：AC006732 ? 372GB_PR3：AC005019 GB_GSS12：AQ390040 ? GB_GSS5：AQ784231 ? ? 2187GB_EST38：AW047296 ? GB_RO：AB004056 ? GB_RO：AB004056 ? 416GB_BA1：SCJ21 ? GB_VI：MCU68299 GB_VI：U93872 ? ? 1827GB_BA1：AFAPHBHI ? GB_PR3：HSJ836E13 ? GB_EST24：AJ170227 ? 390GB_PR3：HUMDODDA GB_PAT：I40899 GB_PAT：I40900 789GB_BA1：SCJ21 ? GB_BA1：BLTRP ? GB_PAT：E01375 ?	4078 ? 17381 159453 ? 188362 680 ? 542 ? ? 614 ? 1581 ? 1581 ? 31717 ? 230278 133661 ? ? 4501 ? 78055 ? 409 ? 26764 26764 1317 31717 ? 7725 ? 7726 ?	AB007125 ? U64861 AC006732 ? AC005019 AQ390040 ? AQ784231 ? ? AW047296 ? AB004056 ? AB004056 ? AL109747 ? U68299 U93872 ? ? M69036 ? AL050326 ? Al170227 ? L39874 I40899 I40900 AL109747 ? X04960 ? E01375 ?	Serratia?marcescens?slaA?gene?for?surface?layerprotein.com?plete cds，isolate?8000. Caenorhabditis?elegans?cosmid?C47D2 Caenorhabditis?elegans?clone?Y32G9. ^＊＊＊SEQUENCING?IN PROGRESS ^＊＊＊.9unordered?pieces. Homo?sapiens?BAC?clone?GS250A16from?7p21-p22，complete RPCI11-157C9.TJ?RPCI-11Homo?sapiens?genomic?clone?RPCI- 11-157C9.genomicsurvey?sequence. HS_3087_B1_C10_T7C?Cl--Approved?Human?Genomic?Sperm Library?D?Homo?sapiens?genomuc?clone?Plale＝3087Col＝19 Row＝F，genomic?suryey?sequence. Ul-M-BH1-amh-e-03-0-Ul.s1?NIH_BMAP_M_S2Mus?musculus cDNA?clone?UI-M-BH1-amh-e-03-0-UI3.mRNAsequence Rattus?norvegicus?mRNA?for?BarH-class?homeodomain transcription?factor.complete?cds. Rattus?norvegicus?mRNA?for?BarH-class?homeodomain transcription?factor.complete?cds. Streptomyces?coelicolor?cosmid?J21. ? Mouse?cytomegalovirus?1complete?genomic?sequence. Kaposi′s?sarcoma-associated?herpesvirus?glycoprotein?M.DNA replication?protein.glycoprotein.DNA?replication?protein.FLICE inhibitory?protein?and?v-cyclin?genes.complete?cds.and?tegument Alcaligenes?eutrophus?protein?H(phbH)and?protein?I(phbI) genes.complete?cds. Human?DNA?sequence?from?clone?836E13?on?chromosome?20 Contains?ESTs.STS?and?GSSs.complete?sequence. EST216152?Normalized?rat?lung.Bento?Soares?Rattus?sp.cDNA clone?RLUCF563end.mRNA?sequence. Homo?sapiens?deoxycytidylate?deaminase?gene.complete?cds. Sequence?1from?patent?US?5622851. Sequence?2from?patent?US?5622851. Streptomyces?coelicolor?cosmid?J21. ? Brevibacterium?lactofermentum?tryptophan?operon. ? DNA?sequence?of?tryptophan?operon. ?	Serratia?marcescens ? Caenorhabditis?elegans Caenorhabditis?elegans ? Homo?sapiens Homo?sapiens ? Homo?sapiens ? ? Mus?musculus ? Rattus?norvegicus ? Rattus?norvegicus ? Streptomyces?coelicolor A3(2) Mouse?cytomegalovirus?1 Kaposi′s?sarcoma- associated?herpesvirus ? Ralstonia?eutropha ? Homo?sapiens ? Rattus?sp. ? Homo?sapiens Unknown. Unknown. Streptomyces?coelicolor A3(2) Corynebacterium glutamicum Corynebacterium glutamicum	40,386 ? 36,207 36,436 ? 39,722 43,137 ? 37,643 ? ? 41,475 ? 41,031 ? 40,717 ? 34,913 ? 40,097 36,029 ? ? 45,624 ? 37,303 ? 39,098 ? 37,644 37,644 37,644 39,398 ? 39,610 ? 46,753 ?	26-MAR-1998 ? 28-Jul-96 23-Feb-99 ? 27-Aug-98 21-MAY-1999 ? 3-Aug-99 ? ? 18-Sep-99 ? 2-Sep-98 ? 2-Sep-98 ? 5-Aug-99 ? 04-DEC-1996 9-Jul-97 ? ? 26-Apr-93 ? 23-Nov-99 ? 20-Jan-99 ? 11-Aug-95 13-MAY-1997 13-MAY-1997 5-Aug-99 ? 10-Feb-99 ? 29-Sep-97 ?

Table 4 (continuing)

rxa?01943

2172GB_BA1：CORPTSMA ? GB_BA1：BRLPTSG ? GB_BA2：AF045481 ?

2656 ? 3163 ? 2841 ?

L18874 ? L18875 ? AF045481 ?

Corynebacterium?glutamicum?phosphoenolpyruvate?sugar phosphotransferase(ptsM)mRNA.com?plete?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 Corynebacterium glutamicum ATCC 13032 full gene group DNA

Corynebacterium glutamicum (ATCC 13032) culture in BHI substratum (Difco), 30 ℃ of thermal agitation overnight incubation.Centrifugal collecting cell is abandoned supernatant, and cell is suspended among the 5ml damping fluid I (all volumes of pointing out of the 5%-of culture original volume all calculate for the 100ml culture volume) again.The composition of damping fluid I: 140.34g/l sucrose, 2.46g/l MgSO ₄* 7H ₂O, 10ml/l KH ₂PO ₄Solution (100g/l, KOH is adjusted to PH6.7), 50g/lM12 enriched material (10g/l (NH ₄) ₂SO ₄, 1g/l NaCl, 2g/l MgSO ₄* 7H ₂O, 0.2g/l CaCl ₂, 0.5g/l yeast extract (Difco)), 10ml/l trace element mixture (200mg/l FeSO ₄* H ₂O, 10mg/l ZnSO ₄* 7H ₂O, 3mg/l MnCl ₂* 4H ₂O, 30mg/l H ₃BO ₃, 20mg/lCoCl ₂* 6H ₂O, 1mg/l NiCl ₂* 6H ₂O, 3mg/l Na ₂MoO ₄* 2H ₂O), 500mg/l complexing agent (EDTA or citric acid), 100ml/l vitamine mixture (0.2mg/l vitamin H, 0.2mg/l folic acid, the amino M-nitro benzoic acid of 20mg/l p-, 20mg/l riboflavin, 40mg/lpanthothenate, 140mg/l nicotinic acid, 40mg/l hydrochloric acid Vitamin B6,200mg/l inositol).Add N,O-Diacetylmuramidase in the suspension to final concentration 2.5mg/ml.37 ℃ hatched about 4 hours after, cell walls is degraded, the centrifugal collection of the protoplastis that obtains.Precipitation is washed once with 5ml damping fluid I, and (10mM Tris-HCl, 1ml EDTA pH8) washes once with 5ml TE damping fluid.Precipitation is resuspended with 4ml TE damping fluid, and adds 0.5ml SDS solution (10%) and 0.5ml NaCl solution (5M).Add Proteinase K to final concentration 200 μ g/ml, suspension was hatched about 18 hours at 37 ℃.DNA extracts purifying with phenol, phenol-chloroform-primary isoamyl alcohol, chloroform-primary isoamyl alcohol according to standard program.Then, add 3M sodium acetate and 2 times of volume of ethanol of 1/50 volume, hatched 30 minutes, with the supercentrifuge 12 that uses SS34 rotary head (Sorvall), centrifugal 30 minutes of 000rpm, deposit D NA at-20 ℃.DNA is dissolved in the 1ml TE damping fluid that contains 20 μ g/ml RNaseA, and 4 ℃ of dialysis are at least 3 hours in 1000ml TE damping fluid.During this period of time, exchange buffering liquid 3 times.In the dna solution of every 0.4ml dialysis, add 0.4ml2M LiCl and 0.8ml ethanol.-20 ℃ hatch 30 minutes after, centrifugal (13,000rpm, Biofuge Fresco, Heraeus, Hanau Germany) collects DNA.The DNA precipitation is melted in the TE damping fluid.DNA by this program preparation can be used for all purposes, comprises the structure of southern hybridization and genomic library.

Embodiment 2: the structure of the genomic library of Corynebacterium glutamicum ATCC13032 in intestinal bacteria

Use the DNA of preparation as described in Example 1, according to the method for known and abundant foundation (referring to, Sambrook for example, J.et al. (1989) " Molecular Cloning:ALaboratory Manual " Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, perhaps Ausubel, F.M.et al. (1994) " Current Protocols inMolecular Bilogy ", John Wiley ﹠amp; Sons.), can make up cosmid library and plasmid library.

Can use any plasmid and clay.Plasmid pBR322 (Sutcliffe, J.G. (1979) Proc.Natl.Acad.Sci.USA, 75:3737-3741); PACY177 (Change ﹠amp; Cohen (1978) J.Bacteriol134:1141-1156), pBS series plasmid (pBSSK+, pBSSK-and other plasmids; Stratagene, LaJolla, USA), clay SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A.and Waterson, R.H. (1987) Gene53:283-286) can be used for special purpose.The special gene library of using in Corynebacterium glutamicum can use plasmid pSL109 (Lee, H-S.and A.J.Sinskey (1994) J.Microbiol.Biotechnol.4:256-263) to make up.

Embodiment 3:DNA order-checking and computer function analysis

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

Embodiment 4: vivo mutations

(for example can pass through intestinal bacteria or other microorganisms, some bacterium of genus bacillus or similarly be the yeast of yeast saccharomyces cerevisiae) the going down to posterity of plasmid (perhaps other carriers) DNA, carry out the vivo mutations of Corynebacterium glutamicum, wherein these microorganisms keep the ability of its genetic information globality to be damaged.Typical mutant strain has sudden change (for example, mutHLS, mutD, mutT etc. in the gene of DNA repair system; Reference is referring to Rupp, W.D. (1996) DNA repair mechanisms, and in:Escherichiacoli and Salmonella, p.2277-2294, ASM:Washington.).These bacterial strains are known for the people who is skilled in technique.The use of these bacterial strains is set forth in, Greener for example, and A.and Callahan is among M. (1994) the Strategies 7:32-34.

Embodiment 5: the DNA that transmits between intestinal bacteria and Corynebacterium glutamicum

Rod bacillus and tyrothricin bacterial classification contain can the spontaneous endogenous plasmid that duplicates (for example similarly being, pHM1519 or pBL1) (comment referring to, for example, Martin, J.F.et al. (1987) Biotechnology, 5:137-146).The shuttle vectors of intestinal bacteria and Corynebacterium glutamicum, can use colibacillary standard vector to be easy to make up (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 ﹠amp; Sons.), promptly add replication fork starting point of Corynebacterium glutamicum and suitable mark therein.This replication origin, what preferably isolating endogenous plasmid obtained from excellent bacillus and tyrothricin bacterial classification.As these these special purposes of bacterial classification transformation marker is kalamycin resistance gene (for example from Tn5 or Tn903 transposon those kalamycin resistance genes) or chloramphenicol resistance gene (Winnacker, E.L. (1987) " From Genes to Clones-Introduction to Gene Technology; VCH, Weinheim).In the document that makes up various wild-type shuttle vectorss, many examples are arranged, these shuttle vectorss can duplicate in intestinal bacteria and Corynebacterium glutamicum, and can be used for various purposes, comprising gene overexpression (reference referring to, for example, 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).

Use the standard method can be in above-mentioned shuttle vectors, and can carry this hybridization and introduce in the Corynebacterium glutamicum strain interested gene clone.The conversion of Corynebacterium glutamicum can be passed through protoplast transformation (Kastsumata, R.et al. (1984) J.Bacteriol.159306-311), fax hole (Liebl, E.et al. (1989) FEMSMicrobiol.Letters, 53:399-303) realize, when using special carrier, also can pass through keying action (for example at Sch  fer, A et al. (1990) J.Bacteriol.172:1663-1666) and realize.Also can be by preparing plasmid DNA (known standard method on the use technology) from Corynebacterium glutamicum and it be transformed into the intestinal bacteria, and shuttle vectors is transferred to intestinal bacteria from Corynebacterium glutamicum.This step of converting can use standard method to carry out, but is to use Mcr defective escherichia coli bacterial strain, for example NM522 (Gough; Murray (1983) J.Mol.Biol.166:1-19) be favourable.

Use contains pCG1 (U.S.Patent No.4,617,267) or its segmental plasmid, and can select kalamycin resistance gene (Grindley from TN903, N.D.and Joyce, C.M. (1980) Proc.Natl.Acad Sci.USA 77 (12): 7176-7180), just can be in Corynebacterium glutamicum the overexpression gene.In addition, use plasmid pSL109 (Lee, H.-S.and A.J.Sinskey (1994) J.Microbiol.Biotechnol.4:256-263) also can be in Corynebacterium glutamicum the overexpression gene.

Except using reproducible plasmid, also can realize the overexpression of gene by genome conformity.The genome conformity of Corynebacterium glutamicum or other excellent bacillus or tyrothricin bacterial classification, can realize by the method for knowing, the homologous recombination of genome area for example, the integration (REMI) of restriction endonuclease mediation is (referring to for example, DE Patent 19823834), perhaps by using transposon.Also can be by (for example modifying regulation domain, promotor, repressor and/or enhanser), by using site-directed method (for example homologous recombination) or based on sequence modification, insertion or the disappearance of random occurrence method (for example transposon mutant or REMI), the activity of regulating gene of interest.Nucleotide sequence as transcription terminator also can be inserted into 3 ' of one or more genes encoding zone of the present invention; Such terminator is known technically, and is described in for example Winnacker, among E.L. (1987) the From Genes to Clones-Introduction to Gene Technology.VCH:Weinheim.

Embodiment 6: the estimation that mutein is expressed

By the active observation of mutein in the transformed host cell, depend on this fact, promptly mutein is expressed with similar quantity in the mode similar to wild-type protein.A kind of useful method of determining mutator gene transcriptional level (quantitative index that is used for the mRNA of gene product translation) be carry out Northern hybridization (reference referring to, for example, Ausubel et al. (1988) CurrentProtocols in Molecular Biology, Wiley:New York), wherein the design be used in conjunction with the primer mark of gene of interest detectable mark (normally radioactive or chemiluminescent) is arranged, thereby, when whole RNA of organism culture are extracted out, run gel electrophoresis, transfer on the stable and with this probe and hatch, the combination of bonding probes and quantity have just been indicated the existence and the quantity of this gene mRNA.This information is the evidence that mutator gene is transcribed degree.Can use several method to prepare whole cell RNAs from Corynebacterium glutamicum, this knows technically, for example is described in Bormann, among E.R.et al. (1992) Mol.Microbiol.6:317-326.

In order to estimate proteinic existence and relative populations by this mRNA translation, can use standard technique, for example Wesstern hybridization (referring to, for example, Ausubel et al. (1988) CurrentProtocols in Molecular Biology, Wiley:New York).In the method, extract whole cell proteins, separate by gel electrophoresis, transferring to similarly is to hatch altogether on the such medium of nitrocellulose and with the probe of desired protein specific combination, for example antibody.This probe is marked with the mark of the chemiluminescent or colorimetric that is easy to detect usually.The existence of the mark that observes and quantity have been indicated the existence and the quantity that appear at the required mutein in the cell.

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

The Corynebacterium glutamicum of genetic modification can be cultivated in synthetic or spontaneous growth substratum.The various growth medium that is used for Corynebacterium glutamicum is known and is to be easy to obtain (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 substratum contain one or more carbon sources, nitrogenous source, inorganic salt, VITAMIN and trace element.Preferred carbon source is a carbohydrate, for example monose, disaccharides or polysaccharide.For example, glucose, fructose, seminose, semi-lactosi, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or Mierocrystalline cellulose all can be used as good carbon source.Also can provide carbohydrate to substratum, for example molasses or other carbohydrate purified by products by complex compound.The mixture that improves different carbon sources also is favourable.Other available carbon sources have alcohol and organic acid, for example methyl alcohol, ethanol, acetate or lactic acid.The nitrogen compound that nitrogenous source is normally organic or inorganic perhaps contains the material of these compounds.Representational nitrogenous source comprises ammonia or ammonium salt, for example NH ₄Cl or (NH ₄) ₂SO ₄, NH ₄OH, nitrate, urea, amino acid or complicated nitrogenous source, for example corn steep liquor, soyflour, soybean protein, yeast extract, meat extract or other.

Can be included in the inorganic salt compound in the substratum, comprise calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper or the iron of hydrochloride, phosphoric acid salt or vitriol.Sequestrant can be added in the substratum, to keep the solution metal ion.Useful especially sequestrant comprises dihydroxyl phenol, similarly is catechol and Protocatechuic Acid, and perhaps organic acid similarly is a citric acid.Substratum typically also contains somatomedin, for example VITAMIN and growth stimulant, and their example comprises vitamin H, riboflavin, thiamines, folic acid, nicotinic acid, pantothenate and pyridoxol.Somatomedin and salt is often from the medium component of complexity, for example yeast extract, molasses, corn steep liquor and other compositions.The definite composition intensive of substratum compound depends on direct experiment, and for each particular case concrete decision.Pass through at textbook " AppliedMicrobiol.Physiology; A Practical Approach " (eds.P.M.Rhodes about the optimized information of substratum, P.F.Stanbury, IRL Press (1997) pp.53-73, ISBN 0 19 9635773) " in.Also can select growth medium there from commercial supplier, similarly be standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or other.

All nutrient media componentses all will be by heating (1.5bar, 120 ℃, 20 minutes) or sterile filtration sterilization.Component can be sterilized together, perhaps if necessary separately sterilization separately.All nutrient media componentses can just add in the beginning of growth, perhaps can select continuity or adding in batches.

Culture condition is determined respectively each experiment.Temperature should be in 15 ℃ to 45 ℃ scopes.It is constant that temperature can keep, and perhaps changes in experiment.The pH of substratum preferably about 7.0, and can keep by the interpolation of damping fluid in the substratum in 5 to 8.5 scopes.At the representational damping fluid of this purpose is potassium phosphate buffer.Synthetic damping fluid, for example MOPS, HEPES, ACES and other also can replace using or using simultaneously.Also can be by adding NaOH or NH in process of growth ₄OH is to keep stable cultivation pH.If using similarly is the so complicated nutrient media components of yeast extract, can reduce the necessity of adding damping fluid, this is because many complex compounds have this fact of very strong surge capability.If use the fermentor cultivation microorganism, also can use ammonia control pH.

Incubation time is usually in several hours to several days scopes.Choosing of this time is in order to allow the product of accumulation maximum in the liquid medium within.The growth experiment of announcing carries out in various containers, and for example microtiter plate, glass test tube, glass shake bottle or the glass of different sizes or the fermentor tank of metal.In order to screen a large amount of clones, microorganism should be cultivated to be had baffle plate or not to have microtiter plate, the glass test tube of baffle plate or shake in the bottle.The preferred 100ml that uses shakes bottle, adds the required substratum of 10% (volume).Shaking bottle should be placed on the shaking table and shake (25 millimeters of amplitudes), velocity range 100-300rpm.Can reduce vaporization losses by keeping moistening air; Perhaps, vaporization losses is carried out the mathematics correction.

If detect the clone of genetic modification, so also should detect not modified contrast clone or contain basic plasmid but without any the contrast clone who inserts.Use is grown in 30 ℃ of cells of hatching on the agar plate, for example CM flat board (10g/l glucose, 2.5g/l NaCl, 2g/l urea, the many peptones of 10g/l, 5g/l yeast extract, 5g/l gravy extract, 22g/l agar, 2M NaOH transfers to pH6.8), inoculation medium is to OD ₆₀₀Value is 0.5-1.5.The inoculation of substratum can perhaps realize by the pre-culture of the liquid that adds this bacterium by introducing the salt suspensioning liquid from the Corynebacterium glutamicum cell of CM flat board.

Embodiment 8: the analyzed in vitro of mutein function

The activity of enzyme and kinetic parameter to be determined at technical be fine foundation.Any to given determination of activity experiment through the enzyme that changes, must be fit to the sp act of wild-type enzyme, this is fully within technology skilful person's ability.About the comment of the summary of enzyme, and about structure, kinetics, principle, method, application with determine the clear and definite details of many enzymic activity examples, can in for example below with reference to document, find: Dixon, 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.

Activity of proteins in conjunction with DNA can be measured by several technical known methods, for example DNA band shift analysis (being also referred to as gel retardation assay).These protein are to the effect of other developed by molecule, can be with reporter gene assay determination (for example be described in Kolmar, H.et al (1995) EMBO is in J.14:3895-3904, and the reference of quoting).The reporter gene test macro is known, and the application in protokaryon and eukaryotic cell all sets up, and use similarly is beta-tilactase, green fluorescent protein and the such enzyme of several other protein.

The active mensuration of protein called membrane transporters matter can be according to for example being described in Gennis, R.B. (1989) " Pores, Channels and Transporters ", in Biomembrane, Molecular Structureand Function, Springer:Heidelberg, p.85-137; 199-234; Those technology among the and 270-322 are carried out.

Embodiment 9: the effect Analysis that mutein is produced for required product

The effect that the Corynebacterium glutamicum genetic modification is produced for required compound (for example amino acid), can estimate like this, i.e. microorganism of having modified by (for example described above those) growth under the conditions suitable, and analyzing increases substratum and/or the cellular component that required product (for example, amino acid) is produced.These analytical technologies are known for skilled routine techniques person, comprise spectroscopic analysis, thin-layer chromatography, various dyeing process, enzymatic means and microbial process, and similarly be 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.

Except mensuration to final tunning, also can analyze other components of the pathways metabolism that is used for required compound production, for example intermediate and by product are with the production efficiency of throughput, output and/or the compound of determining organism.Analytical procedure comprises that nutrient level (for example in the substratum, carbohydrate, hydrocarbon, nitrogenous source, phosphoric acid and other ions) mensuration, the mensuration that biomass is formed and grown, the analysis of the production of the common meta-bolites of biosynthetic pathway, and to producing the mensuration of gas in the fermentation.The standard method of these mensuration outlines at Applied MicrobialPhysiology, A Practical Approach, and P.M.Rhodes and P.F.Stanbury, eds., IRL Press, p.103-163; In and 165-192 (ISBN:0199635773) and the reference quoted thereof.

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

Reclaim required product from the Corynebacterium glutamicum cell or in the supernatant of above-mentioned substratum, can be undertaken by technical known the whole bag of tricks.If required product is not emiocytosis, can use the standard technique lysing cell, for example mechanical force or ultrasonic wave by low-speed centrifugal collecting cell from substratum so.The centrifugal cell debris of removing keeps the supernatant that contains soluble protein and partly is used to be further purified required compound.If product from Corynebacterium glutamicum emiocytosis, is removed cell with low-speed centrifugal so from substratum, keep supernatant and partly be used to be further purified.

The supernatant part that any purification process obtains is carried out chromatography with appropriate resin, and desired molecule is kept by the chromatography resin, and a lot of impurity in the sample are not retained, and perhaps impurity is kept by resin, and sample is not retained.Use identical or different chromatographic resins, can repeat this chromatographic step as required.The person that is familiar with the routine techniques can be very skilled the suitable chromatographic resin of selection, and know these resins for the most effective application of specific molecular to be purified.The product of purifying can be used and filter or ultrafiltration and concentration, and is stored under the temperature of product stability maximum.

Technical known purification process is very many, and aforesaid purification process does not also mean that and only is confined to this.These purification process are described in, Bailey for example, J.E.﹠amp; Ollis, D.F.BiochmicalEngineering Fundamentals is among the McGraw-Hill:New York (1986).

The characteristic of separating compound and purity can technical standard technique be estimated.This comprises high performance liquid chromatography (HPLC), light-splitting method, dyeing process, thin-layer chromatography, NIRS, enzymatic means or microbial process.These analytical procedures have comment in following document: Patek et al. (1994) Appl.Environ.Microbiol.60:133-140; Malakhova et al. (1996) Biotekhnologiya11:27-32; 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 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: analysis of gene sequences of the present invention

The mensuration of percent homology between sequence comparison and the two sequences, it is technical known technology, can use the mathematical operation rule to finish, the algorithm among Karlin and Altschul (1990) the Proc.Natl.Acad.Sci.USA 87:2264-68 for example, this algorithm has modification in Karlin andAltschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-77.This algorithm is incorporated into Altschul, in NBLAST among et al. (1990) J.Mol.Biol.215:403-10 and the XBLAST program (2.0 editions).The search of BLAST Nucleotide can be carried out with the NBLAST program, score=100, and wordlength=12 can obtain and PTS nucleic acid molecule homologous nucleotide sequence of the present invention.The search of BLAST protein can be carried out with the XBLAST program, score=50, and wordlength=3 can obtain and pts protein matter molecule homologous aminoacid sequence of the present invention.For comparison purposes, in order to obtain gapped sequence contrast, can use to be described in Altschul et al., (1997) NucleicAcids Res.25 (17): the GappedBLAST among the 3389-3402.When using BLAST and Gapped blast program, the person that is familiar with the routine techniques knows for the specific sequence to be analyzed parameter of optimizer (for example, XBLAST and NBLAST) how.

Another mathematical operation rule example that is used for the sequence comparison is Meyers and Miller algorithm ((1998) Comput.Appl.Biosci.4:11-17).This algorithm is incorporated in the ALIGN program (2.0 editions), and this program is the part of GCG sequence nucleotide sequence comparison software bag.When using ALIGN program comparing amino acid sequence, can use PAM120 weight residue table, gap length punishment 12, gap punishment 4.Other sequential analysis algorithm also is known technically, comprises ADVANCE and ADAM, is described among Torelli and Robotti (1994) Comput.Appl.Biosci.10:3-5; And FASTA, be described among Pearson and Lipman (1998) P.N.A.S.85:2444-8.

Article two, the per-cent homology between the aminoacid sequence also can use the GAP program in the GCG software package (http://www.gcg.com has to be provided) to realize, use Blosum 62 matrixes or PAM 250 matrixes, gap component 12,10,8,6 or 4, length component 2,3 or 4.Article two, the per-cent homology between the nucleotide sequence can use the GAP program in the GCG software package to realize, uses canonical parameter, for example gap component 50 and length component 3.

Comparative analysis among gene order of the present invention and the Genbank between the sequence, on can use technology known technology carry out (referring to, for example, 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 compares by the method for three steps and the sequence among the Genbank.In the first step, the nucleotide sequence among the relative Genbank of each bar sequence of the present invention is carried out BLASTN analyze (for example, local sequence comparative analysis), keep 500 the highest couplings and do further to analyze.Then these 500 couplings are made FASTA and search (for example, local and global composite sequence comparative analysis is carried out the sequence contrast to the sequence area that limits therein).Next, to three couplings the highest of every gene order of the present invention and FASTA, use the GAP program (use canonical parameter) in the GCG software package to carry out whole world sequence contrast.In order to obtain correct result, from the sequence length that Genbank selects, the method for knowing on the use technology is adjusted to the length of search sequence.The results are shown in the table 4 of this analysis.The result who obtains although it is so, analyze the result who obtains with the independent GAP (whole world) that every gene of the present invention is carried out with respect to every contrast of Genbank, be consistent, but analyze that significantly reduce required computing time with respect to the GAP (whole world) of big database.Do not obtain the correlated sequence of the present invention of the above sequence of cutoff, in table 4, show, lack the sequence comparative information.The person can furtherly understand to be familiar with the routine techniques, and the GAP sequence contrast percent homology under the title of listing in table 4 " %homology (GPA) " is listed with European digital format, and wherein ', ' represents decimal system point.For example, value " 40, the 345 " representative " 40.345% " in these row.

The structure and the operation of embodiment 12:DNA microarray

Sequence of the present invention can also be used for dna microarray (design of DNA array, methods and applications are technical knows, be described in, 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) structure and application.

Dna microarray uses solid or flexible upholder, comprises nitrocellulose, nylon, glass, silicon or other materials.Nucleic acid molecule can be connected the surface in an orderly way.After the appropriate flags, other nucleic acid or nucleic acid mixture can with the fixed making nucleic acid molecular hybridization, mark can be used to monitor and measure the independent strength of signal of determining the area hybridization molecule.Present method allows the relative or absolute quantity of the whole or selected nucleic acid in quantitatively suitable simultaneously nucleic acid samples or the mixture.Therefore, dna microarray allow multiple (as many as 6800 or more) similar expression of nucleic acid analysis (referring to for example, Schena, M. (1996) BioEssays 18 (5): 427-431).

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

Nucleic acid microarray also can pass through as at Wodicka, the original position oligonucleotide of describing among L.et al. (1997) the NatureBiotechnology 15:1359-1367 is synthetic to be made up.By the photograph plate method, exactly determined zone in the matrix can be exposed in the light.Blocking group is a photo-labile, thereby is activated and adds through subject nucleotide, but is covered up and any modification is not carried out in zone that can not see light.Ensuing protection and photoactivation circulation allow determining the synthetic of the different oligonucleotides in position.The zonule that the present invention determines can be synthesized by the solid phase oligonucleotide on microarray.

Appear at the nucleic acid molecule of the present invention in sample or the mixture of ribonucleotides, can with microarray hybridization.Can be according to these nucleic acid molecule of standard method mark.Briefly, nucleic acid molecule (for example, mRNA molecule or dna molecular) can be labeled by combining with isotropic substance or fluorescently-labeled Nucleotide, for example, and in reverse transcription or DNA are synthetic.The hybridization of labeling nucleic acid and microarray have description (for example at Schena, M.et al. (1995) supra; Wodicka, L.et al. (1997), supra; And DeSaizieu A.et al. (1998) is among the supra).The detection of hybrid molecule and quantitatively will be fit to specific bonding mark.Radio-labeling can be detected, and for example, at Schena, describes among M.et al. (1995) supra, and fluorescent mark also can be surveyed, and for example uses the method for Shalon etal. (1996) Gemone Research 6:639-645.

As mentioned above, the application of sequence of the present invention in dna microarray allows different Corynebacterium glutamicum strains or the comparative analysis of other excellent bacillus.For example,, can promote the research that changes based in the bacterial strain of transcribing the figure of branch individually by the nucleic acid array method, and promote to specific and/or required similarly be the evaluation of the such bacterial strain character important function of gene of pathogenic, throughput and pressure tolerance.Equally, use the nucleic acid array technology, also can compare the figure of branch of genetic expression of the present invention in the fermentation reaction process.

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

Gene of the present invention, composition and method can be used to study the interaction and the kinetics of protein colony, are called " proteomics ".Protein of interest matter colony comprises, but be not limited to, the all protein colony of Corynebacterium glutamicum (for example, with the protein colony of other biological body by comparison), under particular surroundings or metabolism condition (for example, fermentation medium and high temperature or low temperature or high pH or low pH) activated those protein, perhaps at particular growth or activated those protein of etap.

Can be with the various technical Analysis protein colonies of knowing, example gel electrophoresis.Cell protein can or extract by for example cracking and obtain, and also can use various electrophoretic techniques separated from one another.SDS-PAGE (SDS-PAGE) isolated protein is to a great extent based on their molecular wt.Isoelectrofocusing polyacrylamide gel electrophoresis (IEF-PAGE) by etc. some point (this has not only reflected aminoacid sequence, and has shown proteinic posttranslational modification) isolated protein.The protein analysis method that another kind is more preferably is that the continuous combination of IEF-PAGE and SDS-PAGE is called the 2-D-gel electrophoresis (at for example Hermann et al. (1998) Electrophoresis 19:3217-3221; Fountoulakis et al. (1998) Electrophoresis 19:1193-1202; Langen et al. (1997) Electrophoresis 18:1184-1192; Among Antelmann et al. (1997) Electrophoresis18:1451-1463 description is arranged).

Can manifest by standard technique with the isolating protein of these methods, for example by dyeing or mark.Suitable dyeing is known technically, comprises that Xylene Brilliant Cyanine G, silver dye or fluorescence dye, for example Sypro Ruby (Molecular Probes).(for example, include radiolabeled amino acid or other protein precursors in the Corynebacterium glutamicum substratum ³⁵The S-methionine(Met), ³⁵The S-halfcystine, ¹⁴The C-labeled amino acid, ¹⁵N-amino acid, ¹⁵NO ₃Perhaps ¹⁵NH ₄ ⁺Perhaps ¹³The C-labeled amino acid), can so that these cells before its protein separation with regard to labelled protein.Similarly, also can use fluorescent mark.Can extract, isolate and separate these labelled proteins according to aforementioned techniques.

With the protein that these technology manifest, can do further to analyze by measuring used dyestuff or mark.The quantity of specified protein can be used for example optical means, carry out quantitatively determining, and can with compare in other the proteinic quantity on the same clotting glue or on other gels.Can pass through for example optics comparison, spectrum analysis, gel images analysis and scanning, perhaps, the protein on the gel be compared by using photographic film or indicating meter.These technology are known technically.

In order to determine the characteristic of specified protein, can use direct sequence to measure or other standard techniques.For example, can use N-and/or C-end amino acid order-checking (for example Edman degraded), and mass spectroscopy (particularly MALDI or ESI technology (referring to for example, Langen et al. (1997) Electrophoresis 18:1184-1192)).Protein sequence provided herein can be as the Corynebacterium glutamicum identification of proteins of being undertaken by these technology.

The information that obtains by these technology, can be used for the various patterns that (for example, the different organisms in other conditions, fermentation time point, culture medium condition or coenocorrelation) different sample rooms are modified under comparison protein existence, activity, the different biotic conditions.The data that these tests obtain, can be independent, what perhaps combine with other technologies is used for various application, the behavior of for example comparing (for example metabolism situation) various organisms under the particular case, increase the throughput of the bacterial strain of producing the fine chemistry material, perhaps increase the efficient of fine chemistry material production.

Be equal to statement

The person can recognize to be familiar with the routine techniques, perhaps can determine only to use normal experiment, and particular of the present invention described herein has a lot of Equivalents.Following claim intention comprises these Equivalents.

Sequence table

＜110〉BASF AG (BASF Aktiengesellschaft)

＜120〉coding phosphoenolpyruvic acid: the proteic Corynebacterium glutamicum gene of carbohydrate phosphotransferase system

<130>BGI-122CPPC

<140>PCT/IB00/00973

<141>2000-06-27

<150>US?60/142,691

<151>1999-07-01

<150>US?60/150,310

<151>1999-08-23

<150>DE?19942095.5

<151>1999-09-03

<150>DE?19942097.1

<151>1999-09-03

<160>36

<210>1

<211>1527

<212>DNA

＜213〉Corynebacterium glutamicum (Corynebacterium glutamicum)

<220>

<221>CDS

<222>(101)..(1504)

<223>RXS00315

<400>1

ctcatggcat?ctgcgccgtt?cgcgttcttg?ccagtgttgg?ttggtttcac?cgcaaccaag?60

cgtttcggcg?gcaatgagtt?cctgggcgcc?gcgtattggt?atg?gcg?atg?gtg?ttc 115

Met?Ala?Met?Val?Phe

1 5

ccg?agc?ttg?gtg?aac?ggc?tac?gac?gtg?gcc?gcc?acc?atg?gct?gcg?ggc 163

Pro?Ser?Leu?Val?Asn?Gly?Tyr?Asp?Val?Ala?Ala?Thr?Met?Ala?Ala?Gly

10 15 20

gaa?atg?cca?atg?tgg?tcc?ctg?ttt?ggt?tta?gat?gtt?gcc?caa?gcc?ggt 211

Glu?Met?Pro?Met?Trp?Ser?Leu?Phe?Gly?Leu?Asp?Val?Ala?Gln?Ala?Gly

25 30 35

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?get?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?ttc?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?get?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?aae?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?crc?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?TyrPhe?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?Ash?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 2l0

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?Asn?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

GaG?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?Set?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

arc?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?Set?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

355 360 355

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?age?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?ValGlu?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?eta 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?IIe

115 120 125

aag?gtc?aac?ggc?aag?aac?gag?taacctggga?tccatgttgc?gca 428

Lys?Val?Asn?Gly?Lys?Asn?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?Set?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 molecule that comprises the nucleotide sequence of SEQ ID NO:17, or its complementary sequence.

2. coding comprises the isolated nucleic acid molecule of polypeptide of the aminoacid sequence of SEQ ID NO:18, or its complementary sequence.

3. coding comprises the natural isolated nucleic acid molecule that has allele variant of polypeptide of the aminoacid sequence of SEQ ID NO:18, or its complementary sequence.

4. isolated nucleic acid molecule comprises the nucleotide sequence of showing at least 50% identity with the complete nucleotides sequence of SEQ ID NO:17, or its complementary sequence.

5. isolated nucleic acid molecule, it comprises the fragment of at least 15 continuous nucleotides of the nucleotide sequence of SEQ ID NO:17, or its complementary sequence.

6. isolated nucleic acid molecule, its coding comprises the polypeptide that the aminoacid sequence of at least 50% identity is arranged with the complete amino acid sequence of SEQ ID NO:18, or its complementary sequence.

7. isolated nucleic acid molecule comprises each nucleic acid molecule and the nucleotide sequence of coding heterologous polypeptide among the claim 1-6.

8. the carrier that comprises each nucleic acid molecule among the claim 1-7.

9. the carrier of claim 8, this carrier is an expression vector.

10. the host cell of the expression vector transfection of claim 9.

11. the host cell of claim 10, wherein this cell is a microorganism.

12. the host cell of claim 11, wherein this cell belongs to Corynebacterium or brevibacterium sp.

13. the host cell of claim 10, the wherein expression of said nucleic acid molecule causes the adjusting of this cell fine chemistry material production.

14. the host cell of claim 14, wherein said fine chemistry material is selected from following material: organic acid, protein source amino acid, nonprotein source amino acid, purine bases and pyrimidine bases, nucleosides, Nucleotide, lipid, saturated and unsaturated fatty acids, glycol, carbohydrate, aromatics, VITAMIN, cofactor, polyketide and enzyme.

15. produce the method for polypeptide, be included in the host cell of cultivating claim 10 in the suitable medium, thereby produce polypeptide.

16. isolated polypeptide comprises the aminoacid sequence of SEQ ID NO:18.

17. isolated polypeptide comprises the natural allele variant that exists of the polypeptide of the aminoacid sequence that contains SEQ ID NO:18.

18. isolated polypeptide, by a kind of nucleic acid molecule encoding, this nucleic acid molecule comprises the nucleotide sequence of showing at least 50% identity with the complete nucleotides sequence of SEQ ID NO:17.

19. isolated polypeptide comprises the aminoacid sequence that at least 50% identity is arranged with the complete amino acid sequence of SEQ ID NO:18.

20. isolated polypeptide comprises the fragment of the polypeptide of the aminoacid sequence that contains SEQ ID NO:18, wherein said polypeptide fragment keeps containing the biologic activity of polypeptide of the aminoacid sequence of SEQ ID NO:18.

21. isolated polypeptide.Comprise aminoacid sequence by the nucleic acid molecule encoding of the nucleotide sequence that comprises SEQ ID NO:17.

22. each isolated polypeptide among the claim 16-21 further comprises the allogeneic amino acid sequence.

23. produce the method for fine chemistry material, comprise the cell of cultivating claim 10, thereby produce the fine chemistry material.

24. the method for claim 23, wherein said method comprise the step that reclaims the fine chemistry material from said culture in addition.

25. the method for claim 23, wherein said cell belongs to Corynebacterium or brevibacterium sp.

26. the method for claim 23, wherein said cell is selected from down group: Corynebacterium glutamicum, man of great strength's rod bacillus, lily hedysarum scoparium bacillus, Corynebacterium acctoacidophlum, vinegar paddy rod bacillus, have a liking for acetyl rod bacillus, produce ammonia rod bacillus, Corynebacterium fujiokense, Corynebacteriumnitrilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, the difference tyrothricin, brevibacterium flavum, the Xi Shi tyrothricin, the ketoisocaproic tyrothricin, Brevibacteriumketosoreductum, brevibacterium, extension brevibacterium, separate the listed bacterial strain of paraffin tyrothricin and table 3.

27. the method for claim 23, the wherein expression of the nucleic acid molecule of said carrier causes the adjusting of this cell fine chemistry material production.

28. the method for claim 23, wherein said fine chemistry material is selected from following material: organic acid, protein source amino acid, nonprotein source amino acid, purine bases and pyrimidine bases, nucleosides, Nucleotide, lipid, saturated and unsaturated fatty acids, glycol, carbohydrate, aromatics, VITAMIN, cofactor, polyketide and enzyme.

29. the method for claim 23, wherein said fine chemistry material is an amino acid.

30. the method for claim 29, wherein said amino acid is selected from following amino acid: Methionin, L-glutamic acid, glutamine, L-Ala, aspartic acid, glycine, Serine, Threonine, methionine(Met), halfcystine, Xie Ansuan, leucine, Isoleucine, arginine, proline(Pro), Histidine, tyrosine, phenylalanine, tryptophane.

31. produce the method for fine chemistry material, comprise and cultivate such cell that the genomic dna of this cell has been inserted among the claim 1-6 each nucleic acid molecule and has changed.

32. the existence or the active method of corynebacterium diphtheriae among the diagnosis experimenter, comprise the existence of the peptide molecule of at least a nucleic acid molecule that detects claim 1-6 among the experimenter or claim 16-21, thus the existence or the activity of corynebacterium diphtheriae among the diagnosis experimenter.

33. contain the host cell of the nucleic acid molecule of SEQ ID NO:17, wherein said nucleic acid molecule is destroyed.

34. contain the host cell of the nucleic acid molecule of SEQ ID NO:17, nucleic acid molecule wherein contains one or more to be modified the nucleic acid of listing sequence at SEQ ID NO:17.

35. contain the host cell of the nucleic acid molecule of SEQ ID NO:17, wherein this nucleic acid molecule regulation domain is modified with respect to this molecule wild-type regulation domain.