DE102004009453A1 - Process for the preparation of L-amino acids using coryneform bacteria - Google Patents

Abstract

The invention relates to a process for the fermentative production of L-amino acids, in particular L-methionine, in which fermenting the desired L-amino acid producing coryneform bacteria, in which one attenuates the regulator AsuR, in particular switches off or expressed at a low level. The invention further relates to these recombinant microorganisms.

Description

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

Dry Compounds with which in particular L-amino acids, vitamins, nucleosides and nucleotides and D-amino acids are found in human medicine, in the pharmaceutical industry, in cosmetics, in the food industry and in animal nutrition application.

numerous These compounds are coryneformer by fermentation of strains Bacteria, in particular Corynebacterium glutamicum, produced. Because of the big one Meaning becomes constant worked on the improvement of the manufacturing process. process improvements can fermentation measures such as emotion and oxygen supply, or the composition of the nutrient media such as the sugar concentration during fermentation, or the work-up to the product form by, for example, ion exchange chromatography or the intrinsic performance characteristics of the microorganism concern yourself.

to Improve the performance characteristics of these microorganisms Methods of mutagenesis, selection and mutant selection applied. In this way receives man tribes, resistant to antimetabolites, e.g. the lysine analog S- (2-aminoethyl) cysteine or the methionine analogues α-methyl-methionine, Ethionine, norleucine, N-acetylnorleucine, S-trifluoromethyl homocysteine, 2-amino-5-heprenoic acid, seleno-methionine, methionine sulfoximine, Methoxine, 1-aminocyclopentane-carboxylic acid or are auxotrophic for regulatory are significant metabolites and produce L-amino acids.

since Several years ago, methods of recombinant DNA technology were also used for strain improvement producing L-amino acid strains of Corynebacterium glutamicum by amplifying single amino acid biosynthesis genes and the effect on L-amino acid production examined.

Object of the invention

The Inventors have set themselves the task of new foundations for improved Process for the fermentative production of L-amino acids, in particular L-methionine to provide with coryneform bacteria.

description the invention

Become in the following L-amino acids or amino acids mentioned, are thus one or more of the protein amino acids including theirs Salts, 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. Especially preferred is L-methionine.

Under proteinogenic amino acids one understands the amino acids, in natural Proteins, that is in proteins of microorganisms, plants, animals and humans occurrence. They serve as structural units for proteins in which they have peptide bonds linked together are.

Become in the following L-methionine or methionine are mentioned, so are the salts such as. Methionine hydrochloride or methionine sulfate meant.

The regulator AsuR is an activator of genes involved in the uptake and utilization of sulfur-containing compounds, in particular sulfonates. He is repressed by sulfate. Furthermore, AsuR represses genes of cysteine biosynthesis. Cysteine biosynthesis is of great importance for methionine biosynthesis, as 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 utili zation regulator ".

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

It It was found that the function of the regulator AsuR from Corynebacterium glutamicum the expression of genes involved in the uptake and recovery sulfur-containing compounds, in particular sulfonates involved are activated and repressed genes of cysteine biosynthesis.

The weaken, in particular turning off the for the regulator AsuR coding asuR gene improves the production of L-methionine in the corresponding coryneform bacteria in the Comparison to the parent organisms without weakening or elimination of this Gene.

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

• a) Fermentation der L-Aminosäure produzierenden rekombinanten coryneformen Bakterien in einem Medium, wobei in diesen das für den Regulator AsuR kodierende Gen asuR abgeschwächt, insbesondere ausgeschaltet ist oder auf niedrigem Niveau exprimiert wird,
• b) Anreicherung der L-Aminosäuren im Medium oder in den Zellen der Bakterien, und
• c) Isolierung der gewünschten L-Aminosäuren, wobei gegebenenfalls Bestandteile der Fermentationsbrühe und/oder der Biomasse in Anteilen (> 0 bis 100 %) oder in ihren Gesamtmengen im Endprodukt verbleiben.

The invention further 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 AsuR gene coding for the regulator AsuR is attenuated, in particular switched off or is expressed at a low level,
• b) accumulation of L-amino acids in the medium or in the cells of the bacteria, and
• c) isolation of the desired L-amino acids, optionally with components of the fermentation broth and / or the biomass remaining in proportions (> 0 to 100%) or in their total amounts in the end product.

The used coryneform bacteria produce preferred already before the weakening or switching off the asuR gene L-amino acids, especially L-methionine.

It was found to be microorganisms, preferably coryneform bacteria after weakening, in particular Turn off the regulator AsuR, in an improved way L-amino acids, in particular L-methionine, produce.

The Nucleotide sequences of said gene of Corynebacterium glutamicum belong to the state of the art and can various patent applications and the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicin (Bethesda, MD, USA).

The Nucleotide sequence of for the regulator AsuR of Corynebacterium glutamicum coding gene can the patent application EP1108790 as Sequence No. 12 and as Sequence no. 1 are removed.

The Nucleotide sequence is also in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) under the Accession Number AX120096 and under the Accession Number AX120085 is stored. Furthermore she is under Accession Number BX927148 from nucleotide 11177 to 10104 of to find the sequence indicated, the amino acid sequence of the associated protein is stored under the Accession Number CAF18575.

The in the given passage described sequence coding for the gene AsuR can be used according to the invention become. Furthermore you can Alleles of the mentioned gene are used, resulting from the degeneration of the genetic code or by functionally neutral sense mutations ( "Sense mutations ").

preferred embodiments can be found in the claims.

The term "attenuation" or "attenuation" in this context describes the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or a gene or allele that codes for a corresponding enzyme with a low activity or the corresponding gene or enzyme or protein inactivated and given if these measures combined.

By the measures the weakening becomes the activity or concentration of the corresponding protein in general 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration the wild-type protein, or the activity or concentration of the protein in the starting microorganism, lowered.

The reduction of the protein concentration is detectable by 1 and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with the corresponding evaluation software in the gel. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by 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 corresponding software for concentration determination (Lohaus and Meyer (1998) Biospektrum 5: 32-39; Lottspeich, Angewandte Chemie 111: 2630-2647 (1999)). The activity of DNA-binding proteins can be measured by DNA band-shift assays (also called gel retardation) as described, for example, in the textbook "Bioanalytics" (Lottspeich / Zorbas, Spektrum Akademischer Verlag Gmbh, Heidelberg, Germany, 1998) and Wilson 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 amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be representative Coryneformer bacteria in particular of the genus Corynebacterium act. In the genus Corynebacterium is in particular the Art Corynebacterium glutamicum, which in the professional world for their ability is known, L-amino acids to produce.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are especially the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium molassecola ATCC17965
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
or such as the L-methionine producing strain
Corynebacterium glutamicum ATCC21608.

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

to Achieving a slowdown can either the expression of the genes or the catalytic or regulatory Properties of the enzyme proteins reduced or eliminated become. If necessary, you can both measures be combined.

Gene expression can be reduced by appropriate culture or by genetic modification (mutation) of the signal structures of gene expression. Signaling structures of gene expression include repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon, and terminators. The skilled artisan, for example, finds this in the patent application WO 96/15246, Boyd and Murphy (Journal of Bacteriology 170: 5949-5952 (1988)), Voskuil and Chambliss (Nucleic Acids Research 26: 3584-3590 (1998)) Pátek et al (Microbiology 142: 1297-309 (1996) and Journal of Biotechnology 104: 311-323 (2003)) and in known textbooks of genetics and molecular biology such as the textbook by Knippers ("Molecular Genetics", 6th Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or Winnacker's ("Gene and Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

One example for the targeted regulation of gene expression is the cloning of the to be attenuated gene under the control of an addition of metered amounts of IPTG (Isopropyl-β-D-thiogalactopyranoside) inducible Promoter such as the trc promoter or the tac promoter. For this vectors such as the Escherichia coli expression vector are suitable pXK99E (WO0226787, filed under the Budapest Treaty on 31. July 2001 in DH5alpha / pXK99E as DSM14440 at the Deutsche Sammlung for microorganisms and cell cultures (DSMZ, Braunschweig, Germany)) or pVWEx2 (Wendisch, Ph. D. thesis, Reports of Forschungszentrum Jülich, Jül-3397, ISSN 0994-2952, Jülich, Germany (1997)), which cloned an IPTG-dependent expression of the Gens in Corynebacterium glutamicum.

used For example, this method has been described in WO0226787 for the regulated expression of the deaD gene by integration of the vector pxK99EdeaD into the genome of Corynebacterium glutamicum and Simic et al. (Applied and Environmental Microbiology 68: 3321-3327 (2002)) for regulated expression of the glyA gene by integration of the vector pK18mobglyA 'in Corynebacterium glutamicum.

A another method for the specific reduction of gene expression is the antisense technique, wherein short oligodeoxy nucleotides or Vectors for the synthesis of longer Antisense RNA can be brought into the target cells. The antisense RNA can be complementary there Bind sections of specific mRNAs and reduce their stability or block the translatability. An example of this can be found the expert in Srivastava et al. (Applied Environmental Microbiology 2000 Oct; 66 (10): 4366-4371).

mutations the change or reduction of the catalytic properties of enzyme proteins to lead, are known in the art; as examples are the Works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (Ph. D. thesis, Reports of Forschungszentrum Jülich, Jül-2906, ISSN09442952, Jülich, Germany (1994)). Summary presentations may be known textbooks genetics and molecular biology e.g. that of Hagemann ("Allgemeine Genetics ", Gustav Fischer Verlag, Stuttgart, 1986).

When Mutations include transitions, transversions, insertions and Deletions into consideration. Dependent on from the effect of amino acid exchange on the enzyme activity is caused by missense mutations ("missense mutations ") or Nonsense mutations ("nonsense mutations "). Insertions or deletions of at least one base pair in one Lead gene to frame shift mutations, as a result wrong amino acids be installed or the translation aborts prematurely. deletions lead by several codons typically to a complete one Failure of enzyme activity. Instructions for producing such mutations belong to State of the art and can known textbooks genetics and molecular biology e.g. 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 Hagemann's ( "General Genetics ", Gustav Fischer Verlag, Stuttgart, 1986).

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

For example, in the method of gene disruption, 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 which are used are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob, pK19mob, pK18mobsacB or pK19mobsacB (Schäfer et al., Gene 145, 69-73 (1994)), pGEM- T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Invitrogen, Groningen, The Netherlands; Shaman (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 pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510-4516). The plasmid vector containing the central region of the coding region of the gene is then converted by conjugation or transformation into the desired strain of C. glutamicum. The method of conjugation is described by Schäfer et al. (Journal of Bacteriology 172: 1663-1666 (1990) and Applied and Environmental Microbiology 60: 756-759 (1994)). Methods for transformation are example as in 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 'or the 5' end. This method has been described, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to eliminate the recA gene of C. glutamicum.

object The invention also includes vectors which are at least 15 25 consecutive nucleotides of the central part of the coding region of the gene included.

at the method of gene replacement becomes a mutation such as. a deletion, insertion or base exchange in the one of interest Gene produced in vitro. The produced allele will turn into one for C. glutamicum is not cloned replicative vector and this subsequently by Transformation or conjugation into the desired host of C. glutamicum. To homologous recombination by means of a first, cross-over event causing integration and a suitable second excision-causing cross-over event in the target gene or in the target sequence to achieve the incorporation of the mutation or of the allele. This method is described by Scharzer and Pühler (Bio / Technology 9: 84-87 (1991) and has been described, for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) was used to delete the pyc gene of C. glutamicum by a deletion turn off or by Wehmeier et al. (Microbiology 144: 1853-1862 (1998)) to paste a deletion in the rel gene of C. glutamicum.

An overview of different Genetic engineering methods for C. glutamicum are given by Kirchner and Tauch (Journal of Biotechnology 104: 287-299 (2003).

In one or more of the genes selected from the group yaeC, abc Uncut can in this way be a deletion, insertion or a Base exchange to be installed.

Farther can it for the production of L-amino acids be beneficial, in addition to mitigate the Regulators AsuR one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, of amino acid export and, where appropriate, either to enhance, in particular to overexpress, regulatory proteins, or mitigate, in particular, turn off or reduce expression.

Of the The term "reinforcement" or "reinforcing" describes in this Related the increase the intracellular activity or concentration of one or more enzymes or proteins in one Microorganism encoded by the corresponding DNA, for example, by increasing the copy number of the gene (s), one strong promoter or a gene or allele used for a corresponding enzyme or protein with a high activity codes and optionally these measures combined.

By the measures the reinforcement, in particular overexpression, becomes the activity or concentration of the corresponding protein in general at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000% based on the wild-type protein or the activity or increased concentration of the protein in the starting microorganism.

The Use of endogenous genes is generally preferred. Under "endogenous Genes "or" endogenous Nucleotide sequences " the genes present in the population of a species Nucleotide sequences.

So can be for example the production of L-methionine in addition to the attenuation of the regulator AsuR one or more of the genes selected amplified from the group of genes or alleles of methionine production, in particular overexpressed become. Under "Genes or alleles of methionine production "are all, preferably endogenous, open reading frames, genes or alleles to understand their amplification / overexpression can improve methionine production.

These include, among others, 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, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A213T. These are summarized and explained in Table 1. Table 1 Genes and alleles of methionine production

Farther can it for the production of L-methionine may be beneficial, in addition to the attenuation of the regulator AsuR simultaneously one or more of the genes selected from the group of genes or alleles responsible for growth or methionine production are not essential to mitigate in particular, turn off or reduce expression.

These include, among others, the following open reading frames, genes or alleles: 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 are summarized and explained in Table 2. Table 2 Genes and alleles that are not essential for methionine production

Finally, can it for the production of amino acids, especially L-methionine, be beneficial, in addition to the attenuation regulator AsuR unwanted To eliminate side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms " in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The produced according to the invention Microorganisms are also the subject of the invention and can be continuous or batchwise in a batch process (batch culturing) or in the fed batch or repeated fed batch process (repetitive feed method) for the purpose of producing L-amino acids become. A summary about known cultivation methods is in the textbook by Chmiel (Bioprozesstechnik 1. Introduction in bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

The to be used culture medium must be in Appropriate to the claims satisfy the respective strains. descriptions of culture media of various microorganisms are described in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington D.C., USA, 1981).

When Carbon source can Sugars and carbohydrates, 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 oil, fatty acids such as. palmitic acid, stearic acid and linoleic acid, Alcohols such as e.g. Glycerine and ethanol and organic acids like e.g. acetic acid be used. These substances can used singly or as a mixture.

When Nitrogen source can 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. The nitrogen sources can used singly or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source. The culture medium must further contain salts of metals such as magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth factors such as amino acids and vitamins can be added to those mentioned above substances are used. In addition, suitable precursors can be added to the culture medium. The said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation.

to pH control of the culture are basic compounds such as sodium hydroxide, Potassium hydroxide, ammonia or ammonia water or acidic compounds like phosphoric acid or sulfuric acid used in a suitable manner. To control foaming can Antifoam agents such as e.g. fatty acid be used. To maintain the stability of plasmids can the medium suitable selective substances such. antibiotics added become. To maintain aerobic conditions, oxygen is used or oxygen-containing gas mixtures such as e.g. Air in the culture entered. The temperature of the culture is usually 20 ° C to 45 ° C, and preferably at 25 ° C up to 40 ° C. The culture is continued until a maximum of the desired Product has formed. This goal is usually within from 10 hours to 160 hours.

With The methods of the invention may be the performance of the bacteria or of the fermentation process with respect to the product concentration ((product per volume), the product yield (formed Product Per Used Carbon Source), Product Formation (Formed Product per volume and time) or other process parameters and Combinations of at least 0.5%, at least 1% or at least 2% be improved.

methods for the determination of L-amino acids are known from the prior art. The analysis can be as well as Spackman et al. (Analytical Chemistry, 30, (1958), 1185-1190) by anion exchange chromatography followed by ninhydrin Derivatization can take place, or it can be done by reversed phase HPLC carried out, as in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174) described. The expert also finds information on this at Ashman et al. (in: Tschesche (eds), Modern Methods in Protein Chemistry. 155-172, de Gruyter, Berlin 1985).

The inventive method is used for the fermentative production of L-methionine.

The Concentration of L-methionine in the final product may optionally be adjusted by the addition of L-methionine to the desired value.

Claims (12)

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 attenuated, in particular switched off or expressed at a low level. Method according to claim 1, characterized in that one produces L-methionine. Process for the fermentative production of L-amino acids, in particular L-methionine according to Claim 1, characterized in that one carries out the following steps: a) Fermentation of the desired L-amino acid producing coryneform bacteria in which the regulator AsuR attenuated especially off or expressed at a low level is; 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, where appropriate, components of the fermentation broth and / or Biomass in its entirety or in proportions (> 0 to 100) remain in the final product. Method according to claim 1 or 3, characterized in that bacteria are used, in which one in addition amplified further genes of the biosynthetic pathway of L-methionine. Method according to claim 1 or 3, characterized in that bacteria are used, in where the metabolic pathways are at least partially eliminated, which reduce the formation of L-methionine. Method according to claim 1 or 3, characterized in that the expression of the (the) Polynucleotide (s) for encoded the regulator AsuR. Method according to claim 1 or 3, characterized in that the regulatory and / or catalytic Properties of the polypeptides (enzyme proteins) for which the Code polynucleotides yaeC, abc and yaeE. Process according to Claim 1 or 3, characterized in that, for the production of L-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 are simultaneously boosted, in particular overexpressed:

A method according to claim 1 or 3, characterized in that one fermented for the production of L-amino acids, in particular L-methionine, coryneform microorganisms in which one simultaneously attenuates one or more of the genes selected from the group listed in Table 2, in particular switches off or the expression decreases:

Method according to one or more of the claims 1-9, by characterized in that one has microorganisms of the species Corynebacterium glutamicum. Recombinant microorganisms, preferably coryneforme Bacteria in which at least that coding for the regulator AsuR Gene weakened, especially off or expressed at a low level is present, compared with the starting organism without attenuation or Shutdown of the gene for the regulator AsuR. Vector containing a fragment of at least 15 consecutive Nucleotides of the sequence (s) of at least one of the genes yaeC, abc and yaeE contains and can not replicate in C. glutamicum.