CN1922322A - Method for producing l-amino acids by means of recombinant coryneform bacteria with reduced activity AsuR regulators - Google Patents

Abstract

The invention relates to a method for producing L-amino acids, in particular L-methyonine by fermentation, wherein coryneform bacteria producing desired L-amino acids are fermented and an AsuR regulator is weaken, in particular deactivated or expressed at a low level. The recombinant bacteria are also disclosed.

Description

Use has the recombinant coryneform bacteria that reduces active AsuR instrumentality and produces the amino acid whose method of L-

The invention provides a kind of bar shaped bacteria that is weakened by the asuR gene wherein of fermenting and produce L-amino acid, the particularly method of L-methionine(Met).Described asuR genes encoding instrumentality AsuR.

Prior art

Some chemical compounds, particularly L-amino acid, VITAMIN, nucleosides and Nucleotide and D-amino acid are used for human medicine and pharmaceutical industry, cosmetic industry, grocery trade and Animal nutrition.

Many these compounds are produced by fermentation of coryneform bacteria bacterial strain, especially Corynebacterium glutamicum (Corynebacterium glutamicum).Because their property of crucial importance has continued to carry out the trial of improved production method.The improvement of production method can relate to the fermentation measure, and as stirring and oxygen supply, the perhaps sugared concentration between the component of nutritional medium such as yeast phase, or the purification process of product is for example passed through ion exchange chromatography, or the intrinsic nature of production of microorganism itself.

For improveing the nature of production of these microorganisms, can use mutagenesis, selection and mutant system of selection.Can obtain metabolic antagonist for example Methionin resemblance S-(2-amino methyl)-halfcystine or methionine(Met) analogue Alpha-Methyl-methionine(Met) with this method, ethionine, nor-leucine, N-acetyl nor-leucine, S-trifluoromethyl homocysteine, 2-amino-5-heprenoiticacid, selenomethionine, methionine(Met) sulfoximine, methoxine, the 1-aminocyclopentanecarboxylic acid resistance or to regulate important metabolite be auxotrophic and produce the amino acid whose bacterial strain of L-.

For a period of time, the method for recombinant DNA technology also has been used to produce the improvement of the amino acid whose Corynebacterium glutamicum strain of L-, and each amino acid bio synthetic gene and research are carried out the effect of L-amino acid production by increasing.

Goal of the invention

The inventor provides and has used the new basis of bar shaped bacteria by the modification method of fermentation producing L-amino-acid, particularly L-methionine(Met).

Invention is described

When hereinafter mentioning L-amino acid, be meant to be selected from one or more following gal4 amino acid, comprise its salt: L-aspartic acid, altheine, L-Threonine, the L-Serine, L-L-glutamic acid, L-glutaminate, L-glycine, the L-L-Ala, L-halfcystine, L-Xie Ansuan, the L-methionine(Met), L-Isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-Histidine, L-Methionin, L-tryptophane, L-arginine and L-proline(Pro).Preferred especially L-methionine(Met).

Gal4 amino acid is meant the amino acid that occurs in natural protein, i.e. the amino acid that occurs in microorganism, plant, animal and human's body protein.They link together by peptide bond as proteinic structure unit.

L-methionine(Met) or the methionine(Met) hereinafter mentioned also refer to its salt, for example methionine hydrochloride or methionine sulfate.

Instrumentality AsuR is the activator that participates in the gene of the picked-up of sulfocompound, especially sulfonate (ester) and utilization.It is prevented by vitriol.AsuR also prevents the halfcystine biosynthesis gene.The halfcystine biosynthesizing is extremely important for the methionine(Met) biosynthesizing, because the needed sulphur of the biosynthesizing of methionine(Met) is from sulphite, and needed halfcystine is from the halfcystine biosynthesizing." asuR " title is from " the replacement sulfur source utilizes instrumentality (alternate sulfur source utilization regulator) ".

The adjusting albumen of regulating other genetic expression be can be by for example being called helix turn helix unit the specific protein structure in conjunction with DNA and therefore can strengthen or those protein of other genetic transcription that weakens.

The function activation that has been found that the instrumentality AsuR of Corynebacterium glutamicum participates in the picked-up of sulfocompound, especially sulfonate and the expression of gene of utilization, and prevents the biosynthetic gene of halfcystine.

Reduction, the asuR gene of especially eliminating coding and regulating thing AsuR are compared with the initial microorganism of not weakening or eliminate this gene in corresponding bar shaped bacteria, have improved the production of L-methionine(Met).

The invention provides a kind of method of using bar shaped bacteria by fermentation producing L-amino-acid, described bar shaped bacteria produced L-amino acid and wherein the asuR gene of coding and regulating albumin A suR weakened, especially be eliminated, perhaps with low expression level.

The present invention further provides a kind of method, wherein carried out following steps by fermentation producing L-amino-acid:

(a) recombinant coryneform bacteria of fermentation producing L-amino-acid in substratum, the asuR gene of coding and regulating thing AsuR is weakened, especially is eliminated or low expression level in the described bacterium;

(b) the L-amino acid in enrichment medium or the bacterial cell; And

(c) the L-amino acid of separate wishing, partly (＞0-100%) or all fermented liquids composition and/or biomass is optional is retained in the end product.

Used bar shaped bacteria had preferably been produced L-amino acid, particularly L-methionine(Met) before the asuR gene is weakened or eliminates.

Have been found that microorganism, preferred bar shaped bacteria in reduction, especially eliminate instrumentality AsuR after, produce L-amino acid in the mode of improvement, especially the L-methionine(Met).

The nucleotide sequence of described Corynebacterium glutamicum gene known in the art, in multiple patent application, can find description to it, at state-run medical library (National Library ofMedicine, Bethesda, MD, USA) (NationalCenter for Biotechnology Information also has description in database NCBI) in NCBI.

The nucleotide sequence of gene of the instrumentality AsuR of coding Corynebacterium glutamicum is found among the patent application EP1108790 shown in the sequence No.12 and sequence No.1.

Described nucleotide sequence also is recorded in state-run medical library, and (in the database of NCBI USA) (NCBI), registration number is AX120096 and AX120085 for Bethesda, MD.The registration number of also finding the 11177-10104 position Nucleotide of described sequence is BX927148, and the aminoacid sequence of respective egg white matter is with registration number CAF18575 preservation.

According to the present invention, can use the sequence of the encoding gene asuR that in reference, describes.Also can use described gene, because genetic code degeneracy or the allelotrope that has justice sudden change to obtain by neutral function.

Embodiment preferred sees claims.

Term in the literary composition " reduction " is meant and reduces or eliminate in the microorganism by active in one or more enzyme of corresponding D NA coding or the proteinic born of the same parents, for example use weak promoter or use coding to have the gene or the allelotrope of low-level active corresponding enzyme, perhaps make corresponding gene or enzyme or protein inactivation, and these measures of optional combination.

By the reduction measure, the activity of respective egg white matter or concentration are compared with wild-type protein or with activity of proteins described in the initial microorganism or concentration and generally are reduced to 0-75%, 0-50%, 0-25%, 0-10% or 0-5%.

The reduction of protein concn can and use suitable evaluation software optics in gel to differentiate that protein concn detects by 1-and 2-fibrillarin matter gel separation method subsequently.The protein gel of preparation bar shaped bacteria and this proteinic general method of discriminating are described by (Electrophoresis, 22:1712-23 (2001)) such as Hermann.Proteinic concentration also can be carried out Western blot hybridization (Sambrook etal., Molecular Cloning:A Laboratory Manual.2 by using the proteinic specific antibody that will verify ^NdEd.Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, (1989)) and determine concentration (Lohaus and Meyer (1998) Biospektrum 5:32-39 with the analysis of appropriate software optical assessment subsequently; Lottspeich, Angewandte Chemie 111:2630-2647 (1999)).The protein-bonded activity of DNA can be used the DNA band migration to analyze (being also referred to as the gel retardation analysis) and measure, as textbook Bioanalytik (Lottspeich/Zorbas, Spektrum Akademischer VerlagGmbH, Heidelberg, Germany, 1998) described, and used by (J.Bacteriol.183:2151-2155 (2001)) such as Wilson.The conjugated protein effect to other genetic expression of DNA can be used reporter gene analytical procedure (Sambrook et al., the MolecularCloning of multiple abundant description; A Laboratory Manual.2 ^NdEd.Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 1989) confirm.

Microorganism provided by the invention can perhaps produce amino acid from glucose, sucrose, lactose, fructose, maltose, molasses, starch, Mierocrystalline cellulose from glycerine and ethanol.Described microorganism can be a bar shaped bacteria, especially the Corynebacterium glutamicum Pseudomonas.For Corynebacterium, what mention especially is the Corynebacterium glutamicum bacterial classification, and known its of those skilled in the art produced the amino acid whose ability of L-.

Suitable Corynebacterium bacterial strain is Corynebacterium glutamicum, especially wild type strain especially.

Corynebacterium glutamicum ATCC13032

Vinegar paddy rod bacillus (Corynebacterium acetoglutamicum) ATCC15806

Corynebacterium acctoacidophlum (Corynebacterium acetoacidophilum) ATCC13870

Corynebacterium melassecola ATCC17965

Corynebacterium thermoaminogenes FERM BP-1539

Brevibacterium flavum (Brevibacterium flavum) ATCC14067

Brevibacterium lactofermentum (Brevibacterium lactofermentum) ATCC13869 reaches

Fork tyrothricin (Brevibacterium divaricatum) ATCC14020

Perhaps, for example produce the bacterial strain of L-methionine(Met):

Corynebacterium glutamicum ATCC21608

Bacterial strain with ATCC title can derive from American type culture collection (American Type Culture Collection, Manassas, VA, USA).Bacterial strain with FERM title can derive from National Institute of Advanced Industrial Scienceand Technology (AIST Tsukuba Central 6,1-1-1 Higashi, Tsukuba Ibaraki, Japan).Described Corynebacterium thermoaminogenes bacterial strain (FERM BP-1539) is at US-A-5, describes in 250,434.

In order to realize reduction, can reduce or eliminate the catalysis or the accommodation property of expression of gene or zymoprotein.But these two kinds of measures of optional combination.

Genetic expression can be by cultivating or signal structure by hereditary change (sudden change) genetic expression reduces in a suitable manner.The signal structure of genetic expression is for example repressor gene, activator gene, operator gene, promotor, attenuator, ribosome bind site, initiator codon and terminator.Those skilled in the art can find this information in following document: for example patent application WO 96/15246, Boyd and Murphy (Journal of Bacteriology170:5949-5952 (1988)), Voskuil and Chambliss (Nucleic Acids Research26:3584-3590 (1998), P á tek et al. (Microbiology 142:1297-309 (1996) and Journal of Biotechnology 104:311-323 (2003)), and known genetics and molecular biology textbook, as Knippers (" Molekulare Genetik ", 6th edition, GeorgThieme Verlag, Stuttgart, Germany, 1995) or Winnacker (" Gene undKlone ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) textbook.

The example that the target of genetic expression is regulated is the gene clone that will weaken under the control of IPTG (sec.-propyl-β-D-thio-galactose pyran-glucoside) inducible promoter by adding doses, and inducible promoters is trc promotor or tac promotor for example.For this reason, suitable carriers for example is intestinal bacteria (Escherichia coli) expression vector pXK99E (WO0226787, be deposited in German microorganisms cultures center (DSMZ as DH5alpha/pXK99E with preserving number DSM14440 July 31 calendar year 2001 according to budapest treaty, Braunschweig, Germany) or pVWEx2 (Wendisch, the Ph.D paper, Berichte des Forschungszentrums J ü lich, J ü l-3397, ISSN 0994-2952, J ü lich, Germany (1997)), it allows cloned genes IPTG-dependency in Corynebacterium glutamicum to express.

This method is used, for example in patent specification WO0226787, by regulating the deaD expression of gene in the genome that carrier pXK99EdeaD is integrated into Corynebacterium glutamicum, reach as described in (Applied and Environmental Microbiology 68:3321-3327 (2002)) such as Simic and regulate the glyA expression of gene in the Corynebacterium glutamicum by carrier pK18mobglyA ' is integrated into.

The another kind of method that specificity reduces genetic expression is an antisense technology, wherein short oligodeoxynucleotide or carrier is imported synthetic long sense-rna in the target cell.Described sense-rna can be in conjunction with the complementary sections of special mRNA, and reduces its stability or blocking-up convertibility.Those skilled in the art can Srivastava et al. (Applied EnvironmentalMicrobiology, Oct 2000; 66 (10): find example 4366-4371) about this respect.

The sudden change that causes the zymoprotein catalytic property to change or reduce known in the art.The example that can mention comprises Qiu and (Bioscience Biotechnology and Biochemistry 61:1760-1762 (1997)) and M_ckel (Ph.D papers such as Goodman (Journal of Biological Chemistry 272:8611-8617 (1997)), Sugimoto, Berichte desForschungszentrums J ü lich, J ü l-2906, ISSN09442952, J ü lich, the work of Germany (1994).General description is found in known genetics and molecular biology textbook, for example Hagemann (" Allgemeine Genetik ", Gustav Fischer Verlag, the textbook of Stuttgart (1986).

Possible sudden change is conversion, transversion, insertion and disappearance.According to the effect of aminoacid replacement, use term " missense mutation " or " nonsense mutation " to enzymic activity.In gene, insert or lack at least one base pair and cause phase shift mutation, cause mixing wrong amino acid or translation premature interruption like this.Lack a plurality of codon typical cases and cause completely losing of enzymic activity.About the guidance that produces this sudden change is known in the art, is found in known genetics and molecular biology textbook, for example Knippers (" Molekulare Genetik ", 6th edition, GeorgThieme Verlag, Stuttgart, Germany, 1995); Winnacker (" Gene undKlone ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann (" Allgemeine Genetik ", Gustav Fischer Verlag, Stuttgart, 1986).

The common method of gene of sudden change Corynebacterium glutamicum is that (Bio/Technology 9, the described gene disruption of 84-87 (1991) (gene disruption) and gene replacement (gene replacement) method for Schwarzer and P ü hler.

In the gene disruption method, major portion (zentraler Teil) clone that for example will study gene coding region advances in the plasmid vector, and described plasmid vector can duplicate in host's (being typically intestinal bacteria), but in Corynebacterium glutamicum reproducible not.Suitable carriers is pSUP301 (Simon et al. for example, Bio/Technology 1,784-791 (1983)), pK18mob, pK19mob, pK18mobsacB or pK19mobsacB (Sch_fer et al., Gene 145,69-73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Invitrogen, Groningen, Netherlands; Shuman (1994) .Journal of Biological Chemistry 269:32678-84; US Patent5,487,993), pCR_Blunt (Invitrogen, Groningen, Netherlands; Bernard etal., Journal of Molecular Biology, 234:534-541 (1993)) or pEM1 (Schrumpf et al., 1991, Journal of Bacteriology 173:4510-4516).Subsequently, the plasmid vector that will contain the main region of described gene coding region moves in the hope bacterial strain of Corynebacterium glutamicum by engaging or transforming.Described method of joining for example Sch_fer etc. (Journal ofBacteriology 172:1663-1666 (1990) and Applied and EnvironmentalMicrobiology 60:756-759 (1994) are described.Method for transformation is for example at (Applied Microbiology and Biotechnology 29 such as Thierbach, (Bio/Technology 7 for 356-362 (1988), Dunicanand Shivnan, 1067-1070 (1989)) and among the Tauch etc. (FEMSMicrobiolgical Letters 123,343-347 (1994)) describe.After passing through " exchange " homologous recombination, the coding region suppressed by vector sequence interruptions of described research gene obtains to lack respectively 3 ' and 5 ' two terminal incomplete allelotrope.(AppliedMicrobiology and Biotechnology 42,575-580 (1994) use this method to eliminate the recA gene of Corynebacterium glutamicum to Fitzpatrick etc.

The present invention provides at least 15 of the major portion that contains the asuR gene coding region, the carrier of preferred 25 continuous nucleotides in addition.

In " gene substitution " method, be external in the research gene, produce for example lack, sudden change that insertion or base replace.The allelotrope that produces is cloned in the carrier that does not into duplicate in Corynebacterium glutamicum successively, move among the Corynebacterium glutamicum host of hope by transforming or engaging subsequently.By first time " exchange " of produce integrating and after causing in the target gene or suitable second time " exchange " of excising in the target sequence carrying out homologous recombination, can obtain to suddenly change or allelic mixing.This method is described in Scharzer and P ü hlerBio/Technology 9:84-87 (1991), and for example (Microbiology 144 by Peters-Wendisch etc., 915-927 (1998) is used for eliminating by disappearance the pyc gene of Corynebacterium glutamicum, and perhaps (Microbiology 144:1853-1862 (1998) is used for inserting disappearance in Corynebacterium glutamicum rel gene by Wehmeier etc.

Kirchner and Tauch (Journal of Biotechnology 104:287-299 (2003)) provide the summary about the multiple genetic engineering method of Corynebacterium glutamicum.

Can mix disappearance, insertion or base in one or more gene that is selected from yaeC, abc and yaeE group by this way replaces.

In addition, except reduction instrumentality AsuR, strengthen, especially cross express or reduction, especially eliminate or reduce research biosynthetic pathway, glycolysis-, replenish one or more enzyme of approach, tricarboxylic acid cycle, pentose phosphate circulation, amino acid output and optionally regulate proteic expression, also be useful for producing L-amino acid.

The increase by activity or concentration in one or more enzyme of corresponding dna encoding or the proteinic born of the same parents described in the microorganism in term in the literary composition " enhancing ", by for example increasing the copy number of one or more gene, use strong promoter or coding to have the active corresponding enzyme of high level or proteinic gene or allelotrope, and these measures of optional combination.

By strengthening, especially crossing and express, the activity of respective egg white matter or concentration are with respect to the common increase at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500% of activity of proteins or concentration in wild-type protein or the initial microorganism, and maximum increases by 1000% or 2000%.

Usually the preferred native gene that uses." native gene " or " endogenous nucleotide sequence " is meant gene or the nucleotide sequence that exists in a population of species.

Therefore, for example for the production of L-methionine(Met), except reduction instrumentality AsuR, can strengthen, especially cross and express gene or allelic one or more gene that is selected from methionine(Met) production." methionine(Met) produce gene or allelotrope " be interpreted as its enhancings/mistakes expression can so that methionine(Met) production improve those, preferred endogenic open reading frame, gene or allelotrope.

These comprise following open reading frame, gene or allelotrope: accBC, accDA, aecD, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, glyA, hom, hom ^FBR, lysC, lysC ^FBR, metA, metB, metE, metH, metY, msiK, opcA, oxyR, ppc, ppc ^FBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwfA213T.These genes are shown in table 1.

Table 1: gene and allelotrope that methionine(Met) is produced

Title	The enzyme or the proteinic title of coding	Reference	Registration number
				accBC	Acyl group-CoA carboxylase EC6.3.4.14	J_ger et al.Archives of Microbiology(1996) 166：76-82 EP1108790； WO0100805	U35023 AX123524 AX066441
accDA	Acetyl-CoA carboxylase EC6.4.1.2	EP1055725 EP1108790 WO0100805	AX121013 AX066443
				aecD	Cystathionine beta-lyase EC4.4.1.8	Rossol et al.，Journal of Bacteriology 174：2968- 2977(1992)	M89931
cstA	The hungry albumin A of carbon	EP1108790 WO0100804	AX120811 AX066109
				cysD	Sulfate adenylyl transferase subunit II EC2.7.7.4 (sulfate adenylyl transferase chainlet)	EP1108790	AX123177
cysE	Serine acetyltransferase EC2.3.1.30	EP1108790 WO0100843	AX122902 AX063961
				cysH	3 '-adenosine phosphate sulfate reduction enzyme (3 '-phosphoadenyl sulfate reductase) EC1.8.99.4 (3 '-adenosine phosphate 5 '-phosphinylidyne sulfate reduction enzyme)	EP1108790 WO0100842	AX123178 AX066001
cysK	Cysteine synthase EC4.2.99.8	EP1108790 WO0100843	AX122901 AX063963
				cysN	Sulfate adenylyl transferase subunit I EC2.7.7.4 (sulfate adenylyl transferase)	EP1108790	AX123176 AX127152
cysQ	Translocator CysQ (translocator cysQ)	EP1108790 WO0100805	AX127145 AX066423
				dps	DNA protected protein (provide protection during protein starvation)	EP1108790	AX127153

eno	Hydratase, phosphoenolpyruvate EC4.2.1.11	EP1108790 WO0100844 EP1090998 Hermann et al.， Electrophoresis 19：3217- 3221(1998)	AX127146 AX064945 AX136862
				fda	Fructose-bis phosphate aldolase EC4.1.2.13	van der Osten et al.， Molecular Microbiology 3：1625-1637(1989)	X17313
gap	Glyceraldehyde-3-phosphate dehydrogenase EC1.2.1.12	EP1108790 WO0100844 Eikmanns et al.，Journal of Bacteriology 174： 6076-6086(1992)	AX127148 AX064941 X59403
				gap2	Glyceraldehyde-3-phosphate dehydrogenase EC1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2)	EP1108790 WO0100844	AX127146 AX064939
gdh	Glutamate dehydrogenase EC1.4.1.4	EP1108790 WO0100844 Boermann et al.， Molecular Microbiology 6：317-326(1992)	AX127150 AX063811 X59404 X72855
				glyA	Glycine/serine hydroxymethylase EC2.1.2.1	EP1108790	AX127146 AX121194
gnd	6-Phosphogluconic dehydrogenase EC1.1.1.44	EP1108790 WO0100844	AX127147 AX121689 AX065125
				hom	Homoserine dehydrogenase EC1.1.1.3	Peoples et al.，Molecular Microbiology 2：63-72 (1988)	Y00546
hom FBR	Feedback resistance (fbr) homoserine dehydrogenase EC1.1.1.3	Reinscheid et al.，Journal of Bacteriology 173：3228- 30(1991)
				lysC	E.C. 2.7.2.4. EC2.7.2.4	EP1108790 WO0100844 Kalinowski et al.， Molecular Microbiology 5：1197-204(1991)	AX120365 AX063743 X57226
lysC FBR	Feedback resistance (fbr) E.C. 2.7.2.4. EC2.7.2.4	See Table 2
				metA	Homoserine acetyltransferase EC2.3.1.31	Park et al.，Molecular Cells 8：286-94(1998)	AF052652
metB	Cystathionine Gamma-lyase EC4.4.1.1 (cystathionine gamma-synthase)	Hwang et al.，Molecular Cells 9：300-308(1999)	AF126953
				metE	Homocysteine methyl transferase	EP1108790	AX127146

	EC2.1.1.14		AX121345
				metH	Homocysteine methyl transferase (vitamin B12 dependency) EC2.1.1.14	EP1108790	AX127148 AX121747
metY	Acetylhomoserine sulfhydrolase	EP1108790	AX120810 AX127145
				msiK	Sugar input albumen (polysaccharide input albumen)	EP1108790	AX120892
opcA	Glucose-6-phosphate dehydrogenase (G6PD) (glucose-6-phosphate dehydrogenase (G6PD) subunit)	WO0104325	AX076272
				oxyR	Transcriptional	EP1108790	AX122198 AX127149
ppc FBR	Feedback resistance Phosphoenolpyruvate carboxylase EC4.1.1.31	EP0723011 WO0100852
				ppc	Phosphoenolpyruvate carboxylase EC4.1.1.31	EP1108790 O′Reagan et al.，Gene 77(2)：237-251(1989)	AX127148 AX123554 M25819
pgk	Phosphoglyceric kinase EC2.7.2.3	EP1108790 WO0100844 Eikmanns，Journal of Bacteriology 174：6076- 6086(1992)	AX121838 AX127148 AX064943 X59403
				pknA	Protein kinase A	EP1108790	AX120131 AX120085
pknB	Protein kinase B	EP1108790	AX120130 AX120085
				pknD	Protein kinase D	EP1108790	AX127150 AX122469 AX122468
pknG	Protein kinase G	EP1108790	AX127152 AX123109
				ppsA	Phosphoenolpyruvate synthase EC2.7.9.2	EP1108790	AX127144 AX120700 AX122469
ptsH	Phosphotransferase system albumen H EC2.7.1.69 (phosphotransferase system composition H)	EP1108790 WO0100844	AX122210 AX127149 AX069154
				ptsI	Phosphotransferase system enzyme I EC2.7.3.9	EP1108790	AX122206 AX127149
ptsM	Glucose specificity phosphotransferase system enzyme II EC2.7.1.69 (glucose-phosphotransferase-system enzyme II)	Lee et al.，FEMS Microbiology Letters 119(1-2)：137-145(1994)	L18874

pyc	Pyruvate carboxylase EC6.4.1.1	WO9918228 Peters-Wendisch et al.， Microbiology 144：915- 927(1998)	A97276 Y09548
				pyc P458S	Pyruvate carboxylase EC6.4.1.1 aminoacid replacement P458S	EP1108790
sigC	Sigma factor C EC2.7.7.6 (the outer function of kytoplasm substitutes sigma factor C, extracytoplasmic function alternative sigma factor C)	EP1108790	AX120368 AX120085
				sigD	RNA polymerase sigma factor D EC2.7.7.6 (the RNA polymerase sigma factor)	EP1108790	AX120753 AX127144
sigE	Sigma factor E EC2.7.7.6 (the outer function of kytoplasm substitutes sigma factor E)	EP1108790	AX127146 AX121325
				sigH	Sigma factor H EC2.7.7.6 (sigma factor S igH)	EP1108790	AX127145 AX120939
sigM	Sigma factor M EC2.7.7.6 (sigma factor S igM)	EP1108790	AX123500 AX127153
				tal	Transaldolase EC2.2.1.2	WO0104325	AX076272
thyA	Thymidylate synthase EC2.1.1.45	EP1108790	AX121026 AX127145
				tkt	Transketolase EC2.2.1.1	Ikeda et al.，NCBI	AB023377
tpi	Triose-phosphate isomerase EC5.3.1.1	Eikmanns，Journal of Bacteriology 174：6076- 6086(1992)	X59403
				zwa1	Growth factor-21 (somatomedin 1)	EP1111062	AX133781
zwf	G-6-P-1-desaturase EC1.1.1.49	EP1108790 WO0104325	AX127148 AX121827 AX076272
				zwf A213T	G-6-P-1-desaturase EC1.1.1.49 aminoacid replacement A213T	EP1108790

In addition, except reduction instrumentality AsuR, reduction simultaneously, especially eliminating or reduce and be selected from one or more expression of gene of producing nonessential gene or allelotrope group for growth or methionine(Met), can be useful for producing the L-methionine(Met).

These comprise following open reading frame, gene or allelotrope: brnQ, ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, metD, metK, pck, pgi, poxB and zwa2.These genes are shown in table 2.

Table 2: produce nonessential gene and allelotrope for methionine(Met)

The gene title	The enzyme or the proteinic title of coding	Reference	Registration number
				brnQ	The carrier proteins of branched-chain amino acid (branched-chain amino acid movement system carrier proteins)	Tauch et al.，Archives of Microbiology 169(4)：303-12(1998) WO0100805 EP1108790	M89931 AX066841 AX127150
ccpA1	Catabolite control albumen (catabolite control albumin A 1)	WO0100844 EP1108790	AX065267 AX127147
				ccpA2	Catabolite control albumen (catabolite control albumin A 2)	WO0100844 EP1108790	AX065267 AX121594
citA	Sensor (sensor) kinase c itA	EP1108790	AX120161
				citB	Transcriptional CitB	EP1108790	AX120163
citE	Citrate lyase EC4.1.3.6	WO0100844 EP1108790	AX065421 AX127146
				ddh	Diaminopimelate dehydrogenase EC1.4.1.16	Ishino et al.，Nucleic Acids Research 15： 3917(1987) EP1108790	S07384 AX127152
gluA	Glutamate transport ATP-is conjugated protein	Kronemeyer et al.， Journal of Bacteriology 177(5)：1152-8(1995)	X81191
				gluB	L-glutamic acid is conjugated protein	Kronemeyer et al.， Journal of Bacteriology 177(5)：1152-8(1995)	X81191
gluC	Glutamate transport permease (glutamate transport system permease)	Kronemeyer et al.， Journal of Bacteriology 177(5)：1152-8(1995)	X81191
				gluD	Glutamate transport permease (glutamate transport system permease)	Kronemeyer et al.， Journal of Bacteriology 177(5)：1152-8(1995)	X81191
luxR	Transcriptional LuxR	WO0100842 EP1108790	AX065953 AX123320
				luxS	Histidine kinase LuxS	EP1108790	AX123323 AX127153

lysR1	Transcriptional LysR1	EP1108790	AX064673 AX127144
				lysR2	Transcriptional activator LysR2 (transcriptional LysR2)	EP1108790	AX123312
lysR3	Transcriptional LysR3	WO0100842 EP1108790	AX065957 AX127150
				menE	O-succinyl-phenylformic acid CoA ligase enzyme EC6.2.1.26 (O-succinyl-phenylformic acid-CoA ligase enzyme)	WO0100843 EP1108790	AX064599 AX064193 AX127144
metD	Transcriptional MetD	EP1108790	AX123327 AX127153
				metK	Methionine(Met)-adenosine-transferring enzyme EC2.5.1.6 (S-adenosylmethionine synthetic enzyme)	WO0100843 EP1108790	AX063959 AX127148
pck	Phosphoenolpyruvate carboxykinase	WO0100844	AJ269506 AX065053
				pgi	G-6-P isomerase EC5.3.1.9	EP1087015 EP1108790	AX136015 AX127146
poxB	Pyruvic oxidase EC1.2.3.3	WO0100844 EP1096013	AX064959 AX137665
				zwa2	Cell growth factor 2 (growth factor-2)	EP1106693 EP1108790	AX113822 AX127146

At last, except reduction instrumentality AsuR, eliminating non-required second order reaction can be useful (Nakayama: " Breeding of Amino Acid Producing Microorganisms " in:Overproductionof Microbial Products for the production of amino acid, especially L-methionine(Met), Krumphanzl, Sikyta, Vanek (eds.) Academic Press, London, UK, 1982).

The present invention also provides microorganism produced according to the invention, but its continuous production or by batch process or fed-batch method or the discontinuous production of repeated fed-batch method L-amino acid.Known cultural method is seen Chmiel (Bioprozesstechnik 1.Einf ü hrung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) textbook) is described.

Used substratum must meet the demand of studying bacterial strain in a suitable manner.See " Manual of Methods for General Bacteriology " (Washington D.C., USA, 1981) of U.S.'s bacteriology meeting (American Society forBacteriology) about the elaboration of the substratum of multiple microorganism.

Spendable carbon source comprises sugar and carbohydrate, for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and Mierocrystalline cellulose, oil ﹠ fat such as soybean oil, Trisun Oil R 80, Peanut oil and Oleum Cocois, lipid acid such as palmitinic acid, stearic acid and linolic acid, alcohol is as glycerine and ethanol, and organic acid such as acetate.These materials can be used alone or as a mixture.

Spendable nitrogenous source comprises organic compounds containing nitrogen such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soyflour and urea, or inorganicization thing such as ammonium sulfate, ammonium chloride, ammonium phosphate, volatile salt and ammonium nitrate.These nitrogenous sources can be used alone or as a mixture.

Spendable phosphorus source comprises phosphoric acid, potassium primary phosphate or dipotassium hydrogen phosphate, or corresponding sodium salt.Substratum also must contain in addition and is required metal-salt such as sal epsom or the ferric sulfate of growing.At last, except above-mentioned substance, can use the essential material of growth such as amino acid and VITAMIN.In addition, suitable precursor can be added in the substratum.Described material can singly be criticized in the form adding substratum or suitably add in the training period.

In order to control the pH value of culture, can suitably use basic cpd such as sodium hydroxide, potassium hydroxide, ammonia or ammoniacal liquor, or acidic cpd such as phosphoric acid or sulfuric acid.Be the generation of control foam, can use for example fatty acid polyglycol ester of foam reducing composition.For keeping the stability of plasmid, can in substratum, add suitable material, for example microbiotic with selectively acting.For keeping aerobic conditions, can in culture, charge into oxygen or contain oxygen gas mixture, for example air.Culture temperature is usually at 20 ℃～45 ℃, preferred 25 ℃-40 ℃.The product that continues to cultivate until hope forms maximum.This purpose reached in the scope at 10～160 hours usually.

Use method of the present invention, form (product that per unit volume and time form) or other method parameter and combination thereof and can improve at least 0.5%, at least 1% or at least 2% about production concentration (product of per unit volume), product output (product that every consumption carbon source forms), product in bacterium or the fermentation process.

The amino acid whose method of definite L-known in the art.Can carry out as (Analytical Chemistry such as Spackmann, 30, (1958), 1185-1190) described anion-exchange chromatography and subsequently triketohydrindene hydrate derivatize perhaps can carry out (Analytical Chemistry (1979) 51:1167-1174) described reversed-phase HPLC such as Lindroth and analyze.Those skilled in the art also can find relevant information in Ashman etc. (155-172, de Gruyter, Berlin 1985 in Tschesche (ed.), Modern Methods in Protein Chemistry).

Method of the present invention is used for the methionine(Met) by fermentative production L-.

The L-concentration of methionine is optional in the end product can be by adding the L-methionine(Met) to desired value.

Claims (12)

1. produce the amino acid whose method of L-by the fermentation recombinant coryneform bacteria, it is characterized in that instrumentality AsuR is weakened in the used bacterium, especially be eliminated or low expression level.

2. the method for claim 1 is characterized in that producing the L-methionine(Met).

Claim 1 pass through fermentation producing L-amino-acid, the especially method of L-methionine(Met), it is characterized in that carrying out following steps:

A) fermentation produces the amino acid whose bar shaped bacteria of L-of wishing, wherein instrumentality AsuR is weakened, especially is eliminated or low expression level;

B) product of wishing in enrichment medium or the bacterial cell; And

C) separate the L-amino acid of wishing, some or all of (＞0-100%) composition and/or the biomass of fermented liquid randomly are retained in the end product.

4. claim 1 or 3 method is characterized in that other gene of L-methionine biosynthesis pathway is strengthened in addition in the used bacterium.

5. claim 1 or 3 method is characterized in that reducing in the used bacterium pathways metabolism to the small part that the L-methionine(Met) forms and are eliminated.

6. claim 1 or 3 method is characterized in that the expression of the polynucleotide of coding and regulating thing AsuR is lowered.

7. claim 1 or 3 method is characterized in that being lowered by the adjusting and/or the catalytic property of polynucleotide yaeC, abc and yaeE encoded polypeptides (zymoprotein).

8. claim 1 or 3 method is characterized in that being selected from one or more gene shown in the table 1 in the bar shaped bacteria of fermentation and being enhanced simultaneously in order to produce L-amino acid, especially L-methionine(Met), especially cross and express: Title The enzyme or the proteinic title of coding accBC Acyl group-CoA carboxylase EC 6.3.4.14 accDA Acetyl-CoA carboxylase EC 6.4.1.2 aecD Cystathionine beta-lyase EC 4.4.1.8 cstA The hungry albumin A of carbon

cysD Sulfate adenylyl transferase subunit II EC 2.7.7.4 (sulfate adenylyl transferase chainlet) cysE Serine acetyltransferase EC 2.3.1.30 cysH 3 '-adenosine phosphate sulfate reduction enzyme EC 1.8.99.4 (3 '-adenosine phosphate 5 '-phosphinylidyne sulfate reduction enzyme) cysK Cysteine synthase EC 4.2.99.8 cysN Sulfate adenylyl transferase subunit I EC 2.7.7.4 (sulfate adenylyl transferase) cysQ Translocator CysQ (translocator cysQ) dps DNA protected protein (provide protection during protein starvation) eno Hydratase, phosphoenolpyruvate EC 4.2.1.11 fda Fructose-bis phosphate aldolase EC 4.1.2.13 gap Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 gap2 Glyceraldehyde-3-phosphate dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) gdh Glutamate dehydrogenase EC 1.4.1.4 glyA Glycine/serine hydroxymethylase EC 2.1.2.1 gnd 6-Phosphogluconic dehydrogenase EC 1.1.1.44 hom Homoserine dehydrogenase EC 1.1.1.3 hom ^FBR Feedback resistance (fbr) homoserine dehydrogenase EC 1.1.1.3 IysC E.C. 2.7.2.4. EC 2.7.2.4 lysC ^FBR Feedback resistance (fbr) E.C. 2.7.2.4. EC 2.7.2.4 metA Homoserine acetyltransferase EC 2.3.1.31 metB Cystathionine Gamma-lyase EC 4.4.1.1 (cystathionine gamma-synthase)

metE Homocysteine methyl transferase EC 2.1.1.14 metH Homocysteine methyl transferase (vitamin B12 dependency) EC 2.1.1.14 metY Acetylhomoserine sulfhydrolase msiK Sugar input albumen (polysaccharide input albumen) opcA Glucose-6-phosphate dehydrogenase (G6PD) (subunit of glucose-6-phosphate dehydrogenase (G6PD)) oxyR Transcriptional ppc ^FBR Feedback resistance Phosphoenolpyruvate carboxylase EC 4.1.1.31 ppc Keto acid carboxylase EC 4.1.1.31 in the phosphoric acid enol pgk Phosphoglyceric kinase EC 2.7.2.3 pknA Protein kinase A pknB Protein kinase B pknD Protein kinase D pknG Protein kinase G ppsA Phosphoenolpyruvate synthase EC 2.7.9.2 ptsH Phosphotransferase system albumen H EC 2.7.1.69 (phosphotransferase system composition H) ptsI Phosphotransferase system enzyme I EC 2.7.3.9 ptsM Glucose-specificity phosphotransferase system enzyme II EC 2.7.1.69 (glucose-phosphotransferase-system enzyme II) pyc Pyruvate carboxylase EC 6.4.1.1 pyc p458S Pyruvate carboxylase EC 6.4.1.1 aminoacid replacement P458S sigC Sigma factor C EC 2.7.7.6 (the outer function of kytoplasm substitutes sigma factor C) sigD RNA polymerase sigma factor D EC 2.7.7.6 (the RNA polymerase sigma factor) sigE Sigma factor E EC 2.7.7.6 (the outer function of kytoplasm substitutes sigma factor E) sigH Sigma factor H

EC 2.7.7.6 (sigma factor S igH) sigM Sigma factor M EC 2.7.7.6 (sigma factor S igM) tal Transaldolase EC 2.2.1.2 thyA Thymidylate synthase EC 2.1.1.45 tkt Transketolase EC 2.2.1.1 tpi Triose-phosphate isomerase EC 5.3.1.1 zwa1 Growth factor-21 (somatomedin 1) zwf G-6-P-1-desaturase EC 1.1.1.49 zwf a213T G-6-P-1-desaturase EC 1.1.1.49 aminoacid replacement A213T

9. claim 1 or 3 method is characterized in that in order to produce L-amino acid, especially L-methionine(Met), be selected from the bar-shaped microorganism of fermentation one or more gene shown in the table 2 by reduction simultaneously, especially be eliminated or reduce expression: The gene title The enzyme or the proteinic title of coding brnQ The carrier proteins of branched-chain amino acid (branched-chain amino acid movement system carrier proteins) ccpA1 Catabolite control albumen (catabolite control albumin A 1) ccpA2 Catabolite control albumen (catabolite control albumin A 2) citA Sensor kinase c itA citB Transcriptional CitB citE Citrate lyase EC 4.1.3.6 ddh Diaminopimelate dehydrogenase EC 1.4.1.16 gluA Glutamate transport ATP-is conjugated protein gluB L-glutamic acid is conjugated protein gluC Glutamate transport permease (glutamate transport system permease)

gluD Glutamate transport permease (glutamate transport system permease) luxR Transcriptional LuxR luxS Histidine kinase LuxS lysR1 Transcriptional LysR1 lysR2 Transcriptional activator LysR2 (transcriptional LysR2) lysR3 Transcriptional LysR3 menE O-succinyl-phenylformic acid CoA ligase enzyme EC 6.2.1.26 (O-succinyl-phenylformic acid-CoA ligase enzyme) metD Transcriptional MetD metK Methionine adenosyltransferase EC 2.5.1.6 (S-adenosylmethionine synthetic enzyme) pck Phosphoenolpyruvate carboxykinase pgi G-6-P isomerase EC 5.3.1.9 poxB Pyruvic oxidase EC 1.2.3.3 zwa2 Cell growth factor 2 (growth factor-2)

10. each method of claim 1-9, the microorganism that it is characterized in that using the Corynebacterium glutamicum bacterial classification.

11. recombinant microorganism, preferred bar shaped bacteria, the initial microorganism of wherein not weakened with the gene of coding and regulating thing AsuR or eliminating is compared, at least the gene of coding and regulating thing AsuR exists with the reduction form, especially exists with the form that is eliminated or the form of low expression level.

12. carrier, it contains a fragment, and described fragment has at least 15 continuous nucleotides of the sequence of at least one in yaeC, abc and the yaeE gene, and described carrier reproducible not in Corynebacterium glutamicum.