EP1756279A1

EP1756279A1 - Method for producing l-amino acids by means of recombinant coryneform bacteria with reduced activity asur regulators

Info

Publication number: EP1756279A1
Application number: EP05700861A
Authority: EP
Inventors: Daniel Koch; Christian RÜCKERT; Jörn Kalinowski; Alfred Pühler; Brigitte Bathe
Original assignee: Degussa GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2004-02-27
Filing date: 2005-01-13
Publication date: 2007-02-28
Also published as: WO2005083082A1; CN1922322A; DE102004009453A1

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

METHOD FOR PRODUCING L-AMINO ACIDS USING RECOMBINANT CORYNEFORMER BACTERIA WITH REDUCED ACTIVITY OF THE ASUR REGULATOR

The invention relates to a process for the fermentative production of L-amino acids, in particular 5 L-methionine, using coryneform bacteria in which the asuR gene is weakened. The asuR gene codes for the regulator AsuR.

State of the art

Chemical compounds, in particular L-0 amino acids, vitamins, nucleosides and nucleotides and D-amino acids, are used in human medicine, in the pharmaceutical industry, in cosmetics, in the food industry and in animal nutrition.

Many of these compounds are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of the great importance, work is constantly being carried out to improve the manufacturing processes. Process improvements can relate to fermentation-related measures such as stirring and supply of oxygen, or the composition of the nutrient media such as the sugar concentration during fermentation, or the processing into the product form by, for example, ion exchange chromatography or the intrinsic performance properties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used to improve the performance properties of these microorganisms. In this way, strains are obtained which are resistant to antimetabolites such as the lysine analogue S- (2-aminoethyl) cysteine or the methionine analogues α-methyl-methionine, ethionine, norleucine, N-acetylnorleucine, S-trifluoromethylhomocysteine, 2 -amino-5- Heprenoitsäure, seleno-methionine, methionine sulfoximine, methoxine, 1-aminocyclopentane carboxylic acid or are auxotrophic for regulatory-important metabolites and produce L-amino acids. For several years, methods of recombinant DNA technology have also been used to improve the strains of Lory amino acid-producing strains of Corynebacterium glutamicum by amplifying individual amino acid biosynthesis genes and examining the effect on L-amino acid production.

Object of the invention

The inventors have set themselves the task of providing new foundations for improved processes for the fermentative production of L-amino acids, in particular L-methionine, with coryneform bacteria.

Description of the invention

If L-amino acids or amino acids are mentioned below, one or more of the protein amino acids including their salts are selected from the group L-aspartic acid, L-asparagine, L-threonine, L-serine, L-

Glutamic acid, L-glutamine, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine , L-tryptophan, L-arginine and L-proline. L-methionine is particularly preferred.

Proteinogenic amino acids are those

Amino acids that are found in natural proteins, i.e. in proteins from microorganisms, plants, animals and humans. They serve as structural units for proteins in which they are linked to one another via peptide bonds. If L-methionine or methionine are mentioned in the following, this also means the salts such as, for example, methionine hydrochloride or methionine sulfate.

The regulator AsuR is an activator of genes that are involved in the uptake and utilization of sulfur-containing compounds, in particular sulfonates. It is repressed by sulfate. AsuR also represses genes of cysteine biosynthesis. Cysteine biosynthesis is of great importance for methionine biosynthesis, since the sulfur required for the biosynthesis of methionine comes from sulfite and cysteine from cysteine biosynthesis. The term "asuR" is derived from "alternate sulfur source utilization regulator".

Regulatory proteins that regulate the expression of other genes are those proteins that can bind to DNA, for example, by means of a specific protein structure called a helix-turn-helix motif and can thus either increase or decrease the transcription of other genes.

It has been found that the function of the regulator AsuR from Corynebacterium glutamicum activates the expression of genes which are involved in the uptake and utilization of sulfur-containing compounds, in particular sulfonates, and represses genes of cysteine biosynthesis.

The weakening, in particular switching off, of the asuR gene coding for the regulator AsuR improves the production of L-methionine in the corresponding coryneform bacteria compared to the starting organisms without weakening or switching off this gene.

The invention relates to a process for the fermentative production of L-amino acids using coryneform bacteria, which in particular already produce L-amino acids and in which that for regulatory protein AsuR-encoding asuR gene is weakened, in particular switched off or is expressed at a low level.

This invention furthermore relates to a process for the fermentative production of L-amino acids, in which the following steps are carried out:

a) fermentation of the L-amino acid-producing recombinant coryneform bacteria in a medium, in which the gene asuR coding for the regulator AsuR is weakened, in particular switched off or expressed at a low level,

b) accumulation of the L-amino acids in the medium or in the cells of the bacteria, and

c) isolation of the desired L-amino acids, components of the fermentation broth and / or the biomass optionally remaining in portions (> 0 to 100%) or in their total amounts in the end product.

The coryneform bacteria used preferably produce L-amino acids, especially L-methionine, even before the asuR gene is weakened or switched off.

It has been found that microorganisms, preferably coryneform bacteria, produce L-amino acids, in particular L-methionine, in an improved manner after weakening, in particular switching off the regulator AsuR.

The nucleotide sequences of the said gene from

Corynebacterium glutamicum is state of the art and can be found in various patent applications and in the database of the National Center for Biotechnology Information (NCBI) and the National Library of Medicine (Bethesda, MD, USA). The nucleotide sequence of the gene coding for the regulator AsuR of Corynebacterium glutamicum can be found in patent application EP1108790 as sequence No. 12 and as sequence No. 1. The nucleotide sequence is also stored in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicin (Bethesda, MD, USA) under the accession number AX120096 and under the accession number AX120085. It can also be found under the accession number BX927148 from nucleotide 11177 to 10104 of the sequence given, the amino acid sequence of the associated protein is stored under the accession number CAF18575.

The sequence described in the text passage coding for the gene asuR can be used according to the invention. Furthermore, alleles of the gene mentioned can be used, which result from the degeneracy of the genetic code or from function-neutral sense mutations (“sense mutations”).

Preferred embodiments can be found in the claims.

In this context, the term “weakening” or “weakening” describes the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism that are encoded by the corresponding DNA, for example by using a weak promoter or a gene or allele used which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme or protein and optionally combines these measures.

The attenuation measures generally reduce the activity or concentration of the corresponding protein to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein, or the activity or concentration of the protein in the starting microorganism, is lowered.

The reduction in the protein concentration can be demonstrated by means of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration using the appropriate evaluation software in the gel. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that of Hermann et al. (Electrophoresis, 22: 1712-23 (2001)). The protein concentration can also be determined by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular cloning: a laboratory manual. 2 ^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and subsequent optical evaluation with appropriate software for concentration determination (Lohaus and Meyer (1998) Biospectrum 5: 32-39; Lottspeicher, Angewandte Chemie 111: 2630-2647 (1999)). The activity of DNA-binding proteins can be measured by means of DNA band shift assays (also referred to as gel retardation) as described, for example, in the textbook “Bioanalytics” (Lottspeicher / Zorbas, Spectrum Academic Publishing House Ginbh, Heidelberg, Germany, 1998) and by Wilson et al. (J. Bacteriol. 183: 2151-2155 (2001)) The effect of DNA-binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay (Sambrook et al., Molecular cloning. a laboratory manual 2 ^nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)..

The microorganisms which are the subject of the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be Representatives of coryneform bacteria, in particular the genus Corynebacterium. In the genus Corynebacterium, the species Corynebacterium glutamicum should be mentioned in particular, which is known in the art for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are particularly the known wild-type strains

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 Brevibacterium flavumvATCC1408bacterium20 Brecci

or such as the L-methionine producing strain

Corynebacterium glutamicum ATCC21608.

Strains labeled "ATCC" can be obtained from the American Type Culture Collection (Manassas, VA, USA). Strains labeled "FERM" can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1 -1 Higashi, Tsukuba Ibaraki, Japan). The aforementioned strain of Corynebacterium thermoaminogenes (FERM BP-1539) is described in US-A-5,250,434.

To achieve an attenuation, either the expression of the genes or the catalytic or regulatory properties of the enzyme proteins can be reduced or switched off. If necessary, both measures can be combined. The gene expression can be reduced by appropriate culture management or by genetic modification (mutation) of the signal structures of the gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters,

Attenuators, riboso binding sites, the start codon and terminators. The person skilled in the art finds information on this e.g. in patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949-5952 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3584-3590 (1998), in Patek et al. (Microbiology 142: 1297-309 (1996) and Journal of Biotechnology 104: 311-323 (2003)) and in well-known textbooks of genetics and molecular biology such as the textbook by Knippers ("Molecular Genetics", 6th edition, Georg Thieme Verlag, Stuttgart , Germany, 1995) or that of Winnacker ("Gene and Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

An example of the targeted regulation of gene expression is the cloning of the gene to be weakened under the control of a promoter which can be induced by adding dosed amounts of IPTG (isopropyl - /? - D-thiogalactopyranoside) such as, for example, the trc promoter or the tac promoter. Vectors such as the Escherichia coli expression vector pXK99E (WO0226787; deposited according to the Budapest Treaty on July 31, 2001 in

DH5alpha / pXK99E as DSM14440 at the German Collection for Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany)) or pVWEx2 (Wendisch, Ph. D. thesis, reports from Forschungszentrum Jülich, Jül-3397, ISSN 0994-2952, Jülich, Germany ( 1997)), which enable IPTG-dependent expression of the cloned gene in Corynebacterium glutamicum.

This method was used, for example, in patent specification WO0226787 for the regulated expression of the deaD gene by integrating the vector pXK99EdeaD into it Genome of Corynebacterium glutamicum and Simic et al. (Applied and Environmental Microbiology 68: 3321-3327 (2002)) for the regulated expression of the glyA gene by integrating the vector pKlδmobglyA 'into Corynebacterium glutamicum.

Another method for specifically reducing> gene expression is the antisense technique, in which short oligodeoxyucleotides or vectors are brought into the target cells for the synthesis of longer antisense RNA. The antisense RNA can bind to complementary sections of specific RNAs and reduce their stability or block translatability. An example of this can be found by the person skilled in the art in Srivastava et al. (Applied Environmental Microbiology 2000 Oct; 66 (10): 4366-4371).

Mutations which lead to a change or a reduction in the catalytic properties of enzyme proteins are known from the prior art; examples include the work of Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugi oto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (Ph. D. thesis, reports by Forschungszentrum Jülich, Jül-2906, ISSN09442952, Jülich, Germany (1994)). Summarizing representations can be found in well-known textbooks of genetics and molecular biology such as that of Hagemann ("General

Genetics ", Gustav Fischer Verlag, Stuttgart, 1986).

Transitions, transversions, insertions and deletions can be considered as mutations. Depending on the effect of the amino acid exchange on the

Enzyme activity is spoken of missense mutations or nonsense mutations. Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, in the result of which incorrect amino acids are incorporated or the translation terminates prematurely. Deletions from multiple codons typically result in complete loss of enzyme activity. Instructions for generating such mutations are state of the art and can be found in well-known textbooks of genetics and molecular biology such as the textbook by Knippers ("Molecular Genetics", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker (" Gene and Clones ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that of Hagemann (" Allgemeine Genetik ", Gustav Fischer Verlag, Stuttgart, 1986).

A common method for mutating C. glutamicum genes is the method of gene disruption ("gene disruption") and gene replacement ("gene disruption") described by Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)). gene replacement ").

In the gene disruption method, for example, a central part of the coding region of the gene of interest is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum. Examples of vectors are PSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pKlδmob, pKl9mob, pKlβmobsacB or pKl9mobsacB (Schäfer et al., Gene 145, 69-73 (1994)), pGEM- T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Invitrogen, Groningen, The Netherlands; Shu an (1994). Journal of Biological Chemistry 269: 32678-84; US Patent 5,487,993), pCR®Blunt (Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510-4516). The plasmid vector, which contains the central region of the coding region of the gene, is then conjugated or transformed into the desired strain of C. glutamicum transferred. The conjugation method is described, for example, by Schäfer et al. (Journal of Bacteriology 172: 1663-1666 (1990) and Applied and Environmental Microbiology 60: 756-759 (1994)). Methods for transformation are described, for example, by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross-over" event, the coding region of the gene in question is interrupted by the vector sequence and two incomplete alleles are obtained, each of which lacks the 3 'and 5' ends. This method was, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to switch off the recA gene from C. glutamicum.

The invention also relates to vectors which contain at least 15, preferably 25 successive nucleotides of the central part of the coding region of the gene asuR.

In the gene replacement method, a mutation such as, for example, a deletion, insertion or base exchange in the gene of interest is produced in vitro. The allele produced is in turn cloned into a vector which is not replicative for C. glutamicum and then cloned by Transformation or conjugation into the desired host of C. glutamicum after homologous recombination by means of a first, integration-causing "cross-over" event and a suitable second, excision-causing "cross-over" event in the target gene or in the The target sequence is achieved by inserting the mutation or the allele.This method is described by Scharzer and Pühler (Bio / Technology 9: 84-87 (1991) and was described, for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) used the to switch off the pyc gene of C. glutamicum by deletion or by Wehmeier et al. (Microbiology 144: 1853-1862 (1998)) for inserting a deletion in the rel gene of C. glutamicum. An overview of various genetic engineering methods in C. glutamicum is given by Kirchner and Tauch (Journal of Biotechnology 104: 287-299 (2003).

In this way, a deletion, insertion or base exchange can be incorporated into one or more of the genes selected from the group yaeC, abc and yaeE.

Furthermore, it can be advantageous for the production of L-amino acids, in addition to the weakening of the regulator AsuR, one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, the amino acid export and possibly regulatory Either to amplify proteins, in particular to overexpress them, or to weaken them, in particular to switch them off or to reduce expression.

In this context, the term “amplification” or “amplification” describes the increase in the intracellular activity or concentration of one or more enzymes or proteins in a microorganism that are encoded by the corresponding DNA, for example by changing the number of copies of the gene or Genes increased, a strong promoter or a gene or all! used, which codes for a corresponding enzyme or protein with a high activity and optionally combines these measures.

The measures of enhancement, in particular overexpression, generally reduce the activity or concentration of the corresponding protein by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500% , up to 1000% or 2000% based on that of the wild type Protein or the activity or concentration of the protein in the starting microorganism increased.

The use of endogenous genes is generally preferred. “Endogenous genes” or “endogenous nucleotide sequences” means the genes or nucleotide sequences present in the population of a species.

For example, in addition to weakening the regulator AsuR, one or more of the genes selected from the group of genes or alleles of methionine production can be amplified, in particular overexpressed, for the production of L-methionine. “Genes or alleles of methionine production” are understood to mean all, preferably endogenous, open reading frames, genes or alleles, the strengthening / overexpression of which can bring about an improvement in methionine production.

These include the following open reading frames, genes or alleles: 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, ptsl, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwal, zwf and zwf A213T. These are summarized and explained in Table 1.

Table 1

Genes and alleles of methionine production

Furthermore, it may be advantageous for the production of L-methionine, in addition to the weakening of the regulator AsuR, to simultaneously weaken, in particular switch off, one or more of the genes selected from the group of genes or alleles which are not essential for growth or methionine production or decrease expression.

These include the following open reading frames, genes or alleles: brnQ, ccpAl, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2, lysR3, menE, ^' metD, etK , pck, pgi, poxB and zwa2. These are summarized and explained in Table 2. Table 2

Genes and alleles that are not essential for methionine production

Finally, it can be advantageous for the production of amino acids, in particular L-methionine, to switch off undesired side reactions in addition to the weakening of regulators AsuR (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek ( eds.), Academic Press, London, UK, 1982).

The microorganisms produced according to the invention are also a subject of the invention and can be cultured continuously or batchwise in the batch process (batch cultivation) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the purpose of producing L-amino acids. A summary of known ones

Cultivation methods is in Chmiel's textbook (Bioprocess Engineering 1. Introduction to Bio Process Engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of the respective strains in a suitable manner. Descriptions of culture media of various microorganisms are contained in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981).

Sugar and carbohydrates such as e.g. Glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as e.g. Soybean oil, sunflower oil, peanut oil and coconut fat, fatty acids. such as. Palmitic acid, stearic acid and linoleic acid, alcohols such as e.g. Glycerin and ethanol and organic

Acids such as Acetic acid can be used. These substances can be used individually or as a mixture.

Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used as the nitrogen source. The nitrogen sources can be used individually or as a mixture.

Phosphoric acid,

Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used. The culture medium must also contain salts of metals such as e.g. Magnesium sulfate or iron sulfate, which are necessary for growth. After all, essential

Growth substances such as amino acids and vitamins can be used in addition to the substances mentioned above. Suitable precursors can also be added to the culture medium. The feedstocks mentioned can be used for culture in the form of a added one-off approach or added in a suitable manner during cultivation.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid are used in a suitable manner to control the pH of the culture. Anti-foam agents such as e.g. Fatty acid polyglycol esters are used. In order to maintain the stability of plasmids, suitable 'selectively acting substances such as e.g.

Antibiotics are added. In order to maintain aerobic conditions, oxygen or gas mixtures containing oxygen, e.g. Air entered the culture. The temperature of the culture is usually 20 ° C to 45 ° C and preferably 25 ° C to 40 ° C. The culture is continued until a maximum of the desired product has formed. This goal is usually achieved within 10 hours to 160 hours.

With the methods of the invention, the performance of the

Bacteria or the fermentation process with regard to the product concentration ((product per volume), the product yield (product formed per carbon source consumed), the product formation (product formed per volume and time) or other process parameters and

Combinations thereof can be improved by at least 0.5%, at least 1% or at least 2%.

Methods for determining L-amino acids are known from the prior art. The analysis can be carried out as in Spackman et al. (Analytical Chemistry, 30, (1958), 1185-

1190) described by anion exchange chromatography with subsequent ninhydrin derivatization, or it can be carried out by reversed phase HPLC, as in Lindroth et al. (Analytical Chemistry (1979) 51: 1167 to 1174) ^• described. The specialist will also find information on this Ashman et al. (in: Tschesche (ed.), Modern Methods in Protein Chemistry. 155-172, de Gruyter, Berlin 1985).

The method according to the invention serves for the fermentative production of L-methionine.

The concentration of L-methionine in the end product can optionally be adjusted to the desired value by adding L-methionine.

Claims

claims

1. A process for the preparation of L-amino acids by fermentation of recombinant coryneform bacteria, characterized in that bacteria are used in which the regulator AsuR is weakened, in particular switched off or expressed at a low level.

2. The method according to claim 1, characterized in that one produces L-methionine.

3. Process for the fermentative production of L-amino acids, in particular L-methionine according to claim 1, characterized in that the following steps are carried out: a) Fermentation of the coryneform bacteria producing the desired L-amino acid, in which the regulator AsuR is weakened, in particular switched off or is expressed at a low level; b) enrichment of the desired product in the medium or in the cells of the bacteria, and c) isolation of the desired L-amino acid, components of the fermentation broth and / or biomass optionally remaining in their entirety or in portions (> 0 to 100%) in the end product ,

4. The method according to claim 1 or 3, characterized in that bacteria are used in which additional genes of the biosynthetic pathway of L-methionine are additionally amplified.

5. The method according to claim 1 or 3, characterized in that bacteria are used in which the metabolic pathways that reduce the formation of L-methionine are at least partially switched off.

6. The method according to claim 1 or 3, characterized in that the expression of the (the) polynucleotide (s) which (the) encodes for the regulator AsuR, is reduced.

7. The method according to claim 1 or 3, characterized in that reducing the regulatory and / or catalytic properties of the polypeptides (enzyme proteins) for which the polynucleotides yaeC, abc and yaeE code.

8. The method according to claim 1 or 3, characterized in that for the production of I. amino acids, in particular L-methionine, coryneform bacteria are fermented, in which one or more of the genes selected from the group listed in Table 1 is simultaneously amplified , especially overexpressed:

Process according to Claim 1 or 3, characterized in that coryneform microorganisms are fermented to produce L-amino acids, in particular L-methionine, in which one or more of the genes selected from the group listed in Table 2 are simultaneously attenuated, in particular switched off or decreased expression:

10. The method according to one or more of claims 1-9, characterized in that microorganisms of the type Corynebacterium glutamicum are used.

11. Recombinant microorganisms, preferably coryneform bacteria, in which at least the gene coding for the regulator AsuR is weakened, in particular switched off or expressed at a low level, compared to the starting organism without weakening or switching off the gene for the regulator AsuR.

12. Vector which contains a fragment with at least 15 consecutive nucleotides of the sequence (s) of at least one of the genes yaeC, abc and yaeE and cannot replicate in C. glutamicum.