WO2005085463A1

WO2005085463A1 - Method for fermentative preparation of l-amino acids by use of recombinant coryneform bacteria

Info

Publication number: WO2005085463A1
Application number: PCT/EP2005/000242
Authority: WO
Inventors: Christian TRÖTSCHEL; Reinhard Krämer; Andreas Burkovski; Brigitte Bathe
Original assignee: Degussa Ag
Priority date: 2004-02-27
Filing date: 2005-01-13
Publication date: 2005-09-15
Also published as: EP1745138B1; DE602005021342D1; CN1922329A; PL1745138T3; ATE468402T1; DK1745138T3; DE102004009454A1; EP1745138A1; ES2346448T3

Abstract

This invention relates to a method for the fermentative preparation of L-amino acids, especially L-methionine in which one ferments recombinant microorganisms producing the wanted L-amino acid, preferably ferments coryneform bacteria, which take up less or no methionine from the surrounding medium, in which one attenuates coding of one or a plurality of the genes selected from the yaeC, abc and yaeE group for the metD2 methionine uptake system, especially deactivates, or expresses at a lower level, and these recombinant microorganisms.

Description

METHOD FOR FERMENTATIVE PREPARATION OF L-AMINO ACIDS BY USE OF RECOMBINANT CORYNEFORM BACTERIA

The object of invention is a method for the fermentative preparation of L-amino acids in particular L- methionine, by use of recombinant microorganisms, preferably coryneform bacteria, which take up little or no methionine from the surrounding medium. In accordance with the invention this is achieved by attenuated, especially deactivation, of the Met2 methionine uptake system, coded through the genes yaeC, abc and yaeE.

STATE OF ART

Chemical compounds, by which are meant in particular L- amino acids, vitamins, nucleosides and nucleotides and D- amino acids find use in human medicine, in the pharmaceutical industry, in cosmetics, in the food industry and in animal feeding.

Numerous of these compounds are prepared by fermentation of microorganisms, preferably from strains of coryneform bacteria, prepared especially from the corynebacterium glutamicum.

Because of the great significance, work has proceeded throughout on improvement of manufacturing methods . Process improvements may involve technical features of the fermentation like, for example, agitation and supply of oxygen or the composition of the culture media such as, for example, the sugar concentration during fermentation or production processing by, for example, ion exchange chromatography or the intrinsic output characteristics of the microorganism itself.

For improvement of the performance characteristics of these microorganisms the methods of mutagenesis, selection and choice of mutants are utilized. In this manner strains that 030303 BT / A 1 2 are resistant to ant i -metabolites , like for example the lysine analog S- ( 2-aminoethyl ) -cysteine or the methionine analogs -methyl -methionine, ethionine, Norleucine, N- acetylnorleucine , S-trif luoromethylhomocysteine, 2-amino-5- lαeprenoit acid, s el eno -methionine , methioninesulfoxamine, methoxine, 1-aminocyclopentane carboxylic acid, or are auxotrophic for regulatory significant metabolites and produce L-amino acids .

For several years recombinant DNA technology has been employed for strain improvement of amino acid producing s trains of corynebacterium glutamicum, in which one amplifies particular amino acid biosynthesis genes and investigates the effect upon the production of L-amino acid.

OBJECT OF THE INVENTION

The inventors have set themselves the obj ect to provide new foundations for improved methods for the fermentative preparation of L-amino acids , especially L-methionine, with microorganisms , preferably with coryneform bacteria .

DESCRIPTION OF THE INVENTION

L-amino acids or amino acids are mentioned below, meaning therewith one or a plurality of the proteinogenic amino acids including their salts , selected from the group L- aspartic acid, L-asparagine, L-threonine, L-serine, L- cjlutamic 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 . Particularly preferred is L-methionine .

By proteinogenic amino acids is understood the amino acids which occur in natural proteins , that is in proteins of microorganisms , plants , animals and humans . They serve as 030303 BT / AL1 3 structural entities for proteins in which they are linked to each other by peptide bonds .

The L-methionine or methionine mentioned below, refer also to the salts like, for example, methionine hydrochloride or methionine sulfate.

The uptake of methionine from the fermentation medium is very significant for the production of methionine, since a possible re-absorption of the excreted product would lower the production rate. The attenuated genes yaeE, abc and yaeC described in this invention, by way of example for corynebacterium glutamicum, code the ATP binding protein ABC and the permease YaeE, which together form the MetD2 methionine uptake system, for the periplas atic binding protein Yaec.

The attenuation, and particularly the deactivation of one or a plurality of the coding genes yaeC, abc and yaeE for the MetD2 methionine uptake system improves the production of L-methionine in the corresponding coryneform bacteria in comparison to the stating organisms without attenuated or deactivation of these genes .

The object of the invention is a method for fermentative preparation of L-amino acids by use of recombinant microorganisms, preferably coryneform bacteria, which in particular already produce L-amino acids that take up less methionine than the starting organisms or no methionine from the surrounding medium, and in which one or a plurality of the MetD2 methionine uptake system yaeC, abc and yaeC coding nucleotide sequence (s), is (are) attenuated or in particular deactivated or expressed at a lower level .

A further object of the invention is a method for the fermentative preparation of L-amino acids in which the following steps are carried out: 030303 BT / AL1 4 (a) Fermentation of the L-amino acid producing recombinant microorganisms, preferably coryneform bacteria in a medium, wherein less methionine than the starting organisms or no methionine is taken up from the surrounding medium, and in which at least one of the yaeE, abc and yaeC coding genes for the MetD2 methionine uptake system is attenuated, and especially deactivated or is expressed at a lower level,

(b) Enrichment of the L-amino acids in the medium or in the cells of the bacteria,

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

The microorganisms that are employed, preferably coryneform bacteria produce L-amino acids, especially L-methionine, preferably already before the attenuating or the deactivation of the uptake of methionine from the su-crounding medium, which for example is to be accomplished by attenuated or deactivation of one or a plurality of the yaeE, abc and yaeC genes .

It was found that microorganisms, preferably coryneform bacteria, that take up little or no methionine from the surrounding medium, which is achieved by attenuating or deactivation of one or a plurality of the yaeE, abc and yaeC coding genes for the MetD2 methionine uptake system, enhance the production of L-amino acids, especially L- methionine.

The nucleotide sequences of the corynebacterium glutamicum genes mentioned are known to the most recent background art and. different patent applications may be cited, along with the data bank of the National Center for Biotechnology 030303 BT / AL1

Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) . yaeC-genes

Designation: Periplasmatic Binding Protein YaeC

Function : Part of the MetD2 Methionine Uptake System

References Sequences 7061 and 709 from EP1108790

Accession No.: AX127145 and AX120793 abc-genes :

Designation : ATP-Binding Protein ABC

Function: Part of the MetD2 Uptake System

Re erences: Sequences 7061, 708, 7060 and 707 from EP1108790; Sequence 363 from WO0100805; Sequence 509 from WOO100844

Accession No. : AX127145; AX120792; AX127144; AX120791; AX066781; AX065383

yaeE-genes :

Designation: Permease YaeE

Function: Part of the MetD2 Methionine Uptake System

References: Sequences 7060, 7061 and 706 from EP0100805; Sequence 365 from WO0100805

Accession No. : AX127144; AX127145; AX120790; AX066783

The sequences coding for the yaeC, abc and yaeE genes described in the given text locations may be used in accordance with the invention. Moreover, allelics of the mentioned gene, which are given through the degenerative 030303 BT / AL1 6 nature of the genetic code or by functionally neutral "sense mutations", may be used.

Preferred embodiments can be found in the Claims .

The term "attenuating" or "attenuate" in this context describes the lowering or deactivation of the intracellular activity of one or a plurality of enzyme systems, or proteins in a microorganism, which are coded by the corresponding DNA, in which for example one uses a weak promoter or a gene or allelic, which codes for a corresponding enzyme with a lower activity or the corresponding gene or enzyme or protein inactivates and if necessary combines these features .

By the steps of attenuating, the activity or concentration of the corresponding proteins is in general lowered from 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.

The information "lesser" relates the same percentages seen in view of the uptake capability of the recombinant microorganisms, in comparison to the starting microorganisms without attenuated or deactivation of the MetD2 methionine uptake system.

The lowering of the protein concentration is detectable by 1- and 2-dimensional common protein separation and subsequent optical identification of the protein concentration in the gel using appropriate evaluation software. A common method for preparation of protein gels for coryneform bacteria and for identification of the proteins is the strategy described in Hermann et al . (Electrophoresis, 22:1712-23 (2001)). The protein 030303 BT / A l 7 concentration can likewise be analyzed by Western-Blot hybridization with a specific antibody for the proving protein (Saitibrook et al . , Molecular Cloning: A Laboratory Manual. 2^nd Ed. Cold Spring Harbor Laboratory Press, NY, (1989)) and subsequent optical interpretation with appropriate software for determination of concentration (Lohaus and Meyer (1998) Biospektrum 5:32-39; Lottspeich, Angewandte Chemie 111: 2630-2647 (1999)). The activity of DNA-binding proteins can be measured using DNA Band Shift Assays (also denoted as gel retardation) as described for example in the textbook "Bioanalytik" (Lottspeich/Zorbas, Spektrum Akademischer Verlag GmbH, Heidelberg, Germany, 1998) and utilized by Wilson et al . (J. Bacteriol . 183: 2151-2155 (2001)) . The action of DNA binding proteins on the expression of other genes can be proved using different well-descri-bed methods of the Reportergen-Assays (Sambrook et al., Molecular Cloning; a Laboratory Manual. 2^nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .

The microorcanisms which are the object of the present invention can produce amino acids from. glucose, saccharose lactose, fructose, maltose, starches, cellulose, or from glycerol and ethanol . Coryneform bacteria, especially the genus corynebacterium can be involved as agent. For the corynebacteitrium genus , corynebacterium glutamicum is to be mentioned, which is known to experts for its ability to produce L-arnino acids .

Suitable st-trains of the corynebacterium genus, especially the corynebacterium type glutamicum are especially the wild strains.

Corynebacte-erium glutamicum ATCC13032

Corynebacte-rium acetoglutamicum ATCC15806

Corynebacte-trium acetoacidophilum ATCC13870 030303 BT / AL1 8 Corynebacterium melassecola ATCC17965

Corynebacterium thermoaminogenes FERM BP-1539

Brevibacterium flavum ATCC14067

Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020

Or as for example the L-methionine producing strain

Corynebacterium glutamicum ATCC21608

Strains with the designation "ATCC" may be obtained from the American Type Culture Collection (Manassas, VA, USA) .

Strains with the designation "FERM" may be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) . The strain of coryne bacterium mentioned thermo amino gene (FERM BP-1539) is described in U.S. Patent 5,250,434.

To obtain a attenuating either the expression of the gene or the catalytic properties of the enzyme proteins can be degraded or deactivated. If necessary both measures can be combine .

The gene expression can be decreased by suitable culture management or by genetic change (mutation) of the signal structures of the gene expression. Signal structures of the gene expression are for example repressor genes, activator genes, operators, promoters, attenuators, ribosome-binding sites, the start codon and terminators. A person skilled in the art can find information for this purpose in patent application WO 96/15246, in the papers by Boyd and Murphy (Journal of Bacteriology 170: 5949-5952 (1988), Vosuil and Chambliss (Nucleic Acids Research 26: 3584-3590 (1998), 030303 BT / AL-1 9 Patek 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, for example, the textbook by Knippers "Moleculare Genetik" (Molecular Genetics) , 6^th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), or that by Winnacker ("Gene und Klone" (Genes and Clones) , VCH Verlagsgesellschaft, Weinheim, Germany (1990)).

An example of the desired regulation of gene expression is the cloning of genes to be attenuated under control of promoters inducible by addition of dosed amounts of IPTG (isopropyl-β-D-thiogalactopyranoside) inducible promoters such as for example the trc promoter or the tac promoter. For this purpose, vectors such as for example the escherichia coli expression vector pXK99E are suitable (WO0226787; filed in accordance with Budapest contract on July 31 2001 as DH5alpha/pXK99E as DSM14440 at the German Association for Microorganisms and Cell Cultures (DSMZ, Braunscliweig, Germany) or pVWEx2 (Wendisch, Ph. D thesis Reports of the Julich Research Center, Jϋl-3397, ISSN 0994-2952, Julich, Germany (1997)), which makes an IPTG-dependent expression of the cloned gene in corynebacterium glutamicum possible.

This method was for example included in patent WO0226787 for the regulated expression of the deaD gene by integration of the vector pXK99EdeaD into the genome of corynebacterium glutamicum and by Simic et al . (Applied and Environmental Microbiology 68: 3321-3327 (2002)) to the regulated expression of the glyA gene by integration of the vector pKlδmobcjlyA into corynebacterium glutamicum.

A further method for the specific lowering of the gene expression is antisense technology, wherein short oligodesoxynucleotides or vectors are employed in the synthesis of longer antisense RNA in the target cells. 030303 BT / AL1 10 There the antisense RNA can bind to complementary segments of specific mR A and lower their stability or block their ability to block the translocator ability. A person skilled in the art can find an example concerning this in Srivastava et al . (Applied Environmental Microbiology, Oct 2000; 66 (10) : 4366-4371) .

Mutations which lead to a change or lowering of the catalytic properties are known to the state of the art; examples are mentioned in the works of Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and mentioned by Mockel (Ph.D thesis, Berichte des Forschungszentrums Julich (Julich Research Center Reports ) , Jul-2906, ISSN09442952 , Julich, Germany (1994) . Summary presentations may be found in known textbooks on genetics and microbiology such as for example that in Hagemann ("Allgemeine Genetik" (General Genetics), Gustav Fischer Verlag, Stuttgart (1986) .

Transitions, transversions, insertions and deletions come into consideration as mutations . In the dependence of the action of amino acid exchange on enzyme activity "missence mutations" or "nonsense mutations" are spoken about.

Insertions or deletions of at least one base pair in a gene lead to "frame shift mutations", as a consequence of which false amino acids are formed or the translation prematurely terminates. Deletions of a plurality of codons lead typically to a complete breakdown of enzyme activity.

Instructions for the production of such mutations are state of the art and can be found in known textbooks of genetics and microbiology such as for example Knippers ("Molekulare Genetik") (Molecular Genetics), 6^th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) , or that by Winnacker ("Gene und Klone" (Genes and Clones), VCH 030303 BT / AL1 11 Verlagsgesellschaft, Weinheim, Germany (1990) , or that by Hagemann ("Allgemeine Genetik" (General Genetics), Gustav Fischer Verlag, Stuttgart (1986) .

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

In the method of gene disruption, for example, a central portion of the coding region of the gene of interest is cloned in a plasmid vector, which in one host (typically E. coli) can replicate, but not in c. glutamicum. Vectors that are worth considering are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pKl8 mob, pKl9mob, pklδmobsacB or pKl9mobsacB (Schafer 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, U.S. Patent 5,487,993, pCRdBlunt (Invitrogen, Groningen, 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 area of the gene coding region, is subsequently converted by conjugation or transformation into the wanted strain of c. glutamicum. The conjugation method is described for example in Schafer et al . (Journal of Bacteriology 172: 1663-1666 (1990) and Applied and Environmental Microbiology 60: 756-759(1994).

Transformation methods are described for example in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988), Duncan and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al . (FEMS Microbiolgical 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 030303 BT / ALl 12 sequence and one obtains two incomplete allelics each of which lacks the 3 ' or the 5 ' ends . This method was used for example by Fitzpatrick et al . (Applied Microbiology and Biotechnology 42, 575-580 (1994) to deactivate the recA- gene of c. glutamicum.

Vectors which contain at least 15 and preferably 25 successive nucleotides of the central portion of the coding region of at least one of the yaeD, abc, or yaeE genes, are likewise a object of the invention

In the "gene replacement" method, a mutation such as for example a deletion, insertion or base replacement is prepared in the gene of interest in vitro. The prepared allelic on the other hand is cloned in a non-replicative vector for c. glutamicum and this is converted subsequently 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 an appropriate second, an excision effecting "cross-over" event in the target gene or in the intended sequence one achieves entrapment of the mutation or of the allelic. This method is described in Scharzer and^" Pύhleir Bio/Technology 9: 84-87 (1991) and was used for example by Peters-Windisch et al. (Microbiology 144, 915-927 (1998), in order to deactivate the pyc gene of c. glutamicum by a deletion, or by Wehmeier et al. (Microbiology 144: 1853-1862 (1998) for the insertion of a deletion in the rel gene of c. glutamicum. irchner and Tauch (Journal of Biotechnology 104: 287-299 (2003)) provide an overview of different genetic methods for c. glutamicum.

In this manner a deletion, insertion or a base exchange can be built into one or a plurality of the genes selected from the yaeC, abc, and yaeE group . 030303 BT / AL1 13 Furthermore, it can be advantageous for the production of L-amino acids, in addition to decreasing the import of methionine from the surrounding medium, to achieve this, in accordance with the invention, by attenuating one or a plurality of the genes selected from the yaeC, abc and yaE group, one or a plurality of enzymes of the respective biosynthesis routes, of glycolysis, of anaplerotik, of the citric acid cycle, of the pentose phosphate cycle of amino acid export and if necessary regulatory proteins, either to strengthen, or attenuate, especially to deactivate or to decrease the expression.

In this connection, the term "strengthening" or "to strengthen" describes the increase in the intracellular activity or concentration of one or a plurality of enzymes or proteins in a microorganism, which are coded by the corresponding DNA in which., for example, the number of copies of the gene or of the gene is increased, uses a strong promoter or a gene or allelic that for a corresponding enzyme or protein codes with a higher activity, and if necessary combines these measures.

By the measures of strengthening, especially over- expression, the activity or concentration of the corresponding protein increases in general by at least 10 %, 25 %, 50 %, 75 %, 100 %, 150 % 200 %, 300 %, 400 % or 500 %, maximal to 1000 % or 2000 % relative to that of the wild- type protein or the activity or concentration of the protein in the starting microorganism.

The use of endogenous genes is in general preferred.

Under "endogenous genes" or "endogenous nucleotide sequences" is understood the type of genes or nucleotide sequences present in the population.

So, for example, for the preparation of L-methionine, in addition to lowering the import of methionine from the 030303 BT / AL1 14 surrounding medium, in accordance with the invention, to achieve by attenuating one or a plurality of the genes, selected from the yaeC, abc, and yaeE group, one or a plurality of the genes chosen from the group of genes or allelics of methionine production is strengthened, especially over-expressed. Under "genes or allelics of methionine production" are altogether, preferably endogenous, open reading frames, genes or allelics to be understood, whose strengthening/over-expression can lead to improvement of methionine production.

For this purpose belong among others the following reading frames, genes or allelics: accBC, accDA, aecD, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dps, eno, fda, gap, gap2, gdh, gnd, glyA, horn, 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 Allelics in Methionine Production

Name Designation of the Coded Reference Access Enzymes or Proteins ion Number accBC Acyl-CoA Carboxyl se Jager et al . U35023 EC 6.3.4.14 Archives of (acyl-CoA carboxylase) Microbiology (1996) 166:76-82 AX1235 EP1108790; 24 WO0100805 AX0664 41 accDA Acetyl-CoA Carboxylase EP1055725 EC 6.4.1.2 EP1108790 AX1210 (acetyl-CoA carboxylase) WO0100805 13 AX0664 43 aecD Cystathionin beta-Lyase Rossol et M89931 EC 4.4.1.8 al . , Journal (cystathionine beta-lyase) of Bacteriology 174:2968-2977 030303 BT / ALl 15 (1992

030303 BT / ALl 16

030303 BT / ALl 17

030303 BT / ALl 18

030303 BT / ALl

19

030303 BT / ALl 20

030303 BT / ALl 21

Moreover, it can be advantageous for L-methionine production, in addition to the lowering of the import of methionine from the surrounding medium, to achieve it >y attenuated one or a plurality of the genes selected from the yaeC, abc and yaeE group, at the same time to attenuate one or a plurality of the genes selected from the group of genes or allelics, which are not essential for the growth or production of methionine, and especially to deactivate or to lower the expression.

The following open reading frames belong among others for this purpose: brnQ, ccpAl, ccpA2, citA, citB, citE, ddϊi, gluA, gluB, gluC, gluD, luxR, luxS, lysRl, lysR2 , lysR3 , menE, metD, met , pck, pgi, poxB and zwa2. These are summarized and explained in Table 2.

Table 2

Genes and Allelics, which are not Essential for Methionine Production

030303 BT / ALl 22

030303 BT / ALl 23

Finally, it can be advantageous for the production of amino acids, especially L-methionine, in addition to decreasing the import of methionine from the surrounding medium, for 030303 BT / ALl 24 example, to accomplish this by attenuating one or a plurality of the genes, selected from the yaeC, abc and yaeE group, to disable undesirable si-de reactions (Nakayama: "Breeding of Amino Acid Prroducing Microorganisms" in: Overproduction off Microbial Products, Krumphanzl, Sikyta, Vanek (eds.) Acaclemic Press, London, UK, 1982) .

The microorganisms prepared in accorc-lance with the invention are likewise a object of the invention and can be cultivated continuously or discontinuxously in batch processes or in fed batch or repeate-3. fed batch processes for the purpose of production of L-ainino acids . A summary of known cultivation methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfϋhrung in die Bioverfahrenstechnik) , (Bioprocess Technology 1. Introduction to Bioprocess Technology) (Gustav Fischer Verlag, Stuttgart (1991) or in Lehrbixch von Storhas, Bioreactoren und Periphere Einrichturxgen (Textbook by Storhas, (Bioreactors and Ancillary Equipment) (Viewweg Publishers, Braunschweig/Wiesbaden (1994))

The culture medium to be used must satisfy in a suitable manner the claims of the appropriate strains. Descriptions of culture media of different microorganisms are contained in the handbook "Manual of Methods fo r General Bacteriology" of the American Society for Bacteriology (Washington D.C. , USA, (1981)).

As source of carbon, sugars and carbo-hydrates such as glucose, saccharose, lactose, fructos e, maltose, molasses., starch and cellulose, oils and fats siich as soy oil, sunflower oil, groundnut oil and coconut oil , fatty acids such as palmitic acid, stearic acid a-nd linoleic acid, alcohols such as glycerol and ethanol and organic acids such as acetic acid are used. These m-aterials can be used alone or as mixtures . 030303 BT / ALl 25 As sources of nitrogen, organic nitrogen containing compounds such as peptone, yeast extract, meat extract, malt extract, maize water, soybean flour and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used.

As source of phosphorus, phosphoric acid, potassium hydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salts can be used. The culture medium must furthermore contain metal salts which are needed for growth such as, for example, magnesium sulfate or iron sulfate. Finally, in addition to the materials mentioned above, growth materials such as amino acids and vitamins are added. Suitable precursors may also be added to the culture medium. The additive materials may be added to the culture in the form of single charges or be fed in a suitable way during the process of culturing.

For control of the culture's pH, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulfuric acid can be employed in a suitable manner. For control of foaming, antifoaming agents such as for example fatty acid polyglycol esters are employed. To maintain plasmid stability, selectively acting materials such as antibiotics may be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such as air may be fed into the culture. The temperature of the culture is normally between 20 and 45 °C, and preferably between 25 and 40 °C. The culturing process is continued until a maximum amount of the desired product has formed.

This target will normally be achieved within 10 to 160 hours . 030303 BT / ALl 26

With the methods of the invention, the output of the bacteria or of the fermentation process in relation to the product concentration (product per unit volume) , the product yield (product formed per utilized carbon source) , the product formation (product formed per unit volume an time) or other process parameter and combinations thereof can be improved by at least 0.5 %, at least 1 %, or at least 2 %.

Methods for determination of L-amino acids are known to the art. The analyses can be done as described in Spackmann et al. (Analytical Chemistry, 30, (1958), 1185-1190) by anion exchange chromatography with subsequent Ninhydrin derivatization, or they can be done by reversed phase HPLC as described in Lindroth et al . (Analytical Chemistry (1979) 51: 1167-1174). Those skilled in the art will also find information on this in Ashman et al . (in Tschesche (Hrsg) , Modern Methods in Protein Chemistry, 155-172, de Gruyter, Berlin 1985) .

The process in accordance with the invention serves for the production of L-methionine by fermentation.

The concentration of L-methionine in the end product can if necessary be approached by addition of L-methionine to the wanted level .

Claims

030303 BT / ALl 27

1. A method for preparation of L-amino acids by fermentation of recombinant microorganisms, preferably coryneform bacteria, characterized in that bacteria are employed, which take up less methionine than the starting microorganism, or no methionine from the surrounding medium, and in which one or a plurality of the genes yaeC, abc and yaeE, coding for the MetD2 methionine absorption system is (are) attenuated, deactivated or expressed at a lower level .

2. The method in accordance with Claim 1, characterized in that L-methionine is prepared.

3. The process for fermentative preparation of L-amino acids, especially L-methionine in accordance with Claim 1, in that one carries out the following steps:

(a) Fermentation of the wanted L-amino acid producing microorganisms, preferably coryneform bacteria, which take up less methionine than the starting organism or take up no methionine from the surrounding medium, in which one attenuates one or more of the genes selected from the yaeC, abc, and yaeE group, coding for the MetD2 methionine take-up system, attenuates, especially deactivates or expresses at a lower level;

(b) Enrichment of the wanted product in the medium or in the bacteria cells, and

(c) Isolation of the wanted L-amino acid, wherein optionally, components of the fermentation broth and/or biomass remain in their totality or in portions (> 0 to 100) in the end product. 030303 BT / ALl 28

4. The method in accordance with Claims 1 or 3 , characterized in that one employs bacteria in which one additionally strengthens other genes of the biosynthesis route of L-methionine.

5. The method in accordance with Claims 1 or 3 , characterized in that bacteria are employed in which the metabolic pathways are at least partly deactivated, which decrease the formation of L- methionine.

6. The method in accordance with Claims 1 or 3 , characterized in that one lowers the expression of the polynucleotide(s) , which codes (code) for components of methionine uptake from the surrounding medium, for instance one or more of the genes, selected from the yaeC, abc and yaeE group, coding for the methionine uptake system.

7. The method in accordance with Claims 1 or 3 , characterized in that one decreases the regulating and/or catalytic properties of the polypeptides (enzyme proteins), for which the yaeC, abc and yaeE polynucleotides code .

8. The method in accordance with Claims 1 or 3 , characterized in that for the preparation of L-amino acids, especially L-methionine, microorganisms, preferably fermented coryneform bacteria, in which, at the same time one strengthens, especially over- expresses, one or a plurality of the genes selected from the group listed in Table 1:

03 BT / ALl 29 (acetyl-CoA carboxylase) aecD Cystathionin beta-Lyase EC 4.4.1.8 (cystathionine beta-lyase) cstA Carbon Starvation Protein A carbon starvation protein A) cysD Sulfat-Adenylyltransferase Untereinheit II EC 2.7.7.4 (sulfate adenylyltransferase small chain) cysE Serinacetyltransferase EC 2.3.1.30 (serine acetyltransferase) cysH 3 ' -Phosphoadenylsulfat Reduktase EC 1.8.99.4 (3 ' -phosphoadenosine 5'- phosphosulfate reductase) cysK Cystein-Synthase EC 4.2.99.8 (cysteine synthase) cysN Sulfat-Adenylyltransferase Untereinheit I EC 2.7.7.4 (sulfate adenylyltransferase) cysQ Transportprotein CysQ (transporter cysQ) dps DNA-Protection Protein (protection during starv-ation protein) eno Enolase EC 4.2.1.11 (enolase) fda Fruktose Bisphosphat Aldolase EC 4.1.2.13 (fructose bisphosphate aldolase) gap Glyceraldehyd-3-Phosphat Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase) gap2 Glyceraldehyd-3-Phosphate Dehydrogenase EC 1.2.1.12 (glyceraldehyde-3-phosphate dehydrogenase 2) gdh Glutamat Dehydrogenase EC 1.4.1.4 (glutamate dehydrogenase) glyA Glycine/Serin Hydroxymethyltransferase EC 2.1.2.1 (glycine/serine 03 BT / ALl 30

3 BT / ALl 31

3 BT / ALl 32

The method in accordance with Claims 1 or 3 , characterized in that for the preparation of L-amino acids, especially L-methothionine, one ferments microorganisms, preferably coryneform microorganisms, in which at the same time one attenuates, especially deactivates or decreases the expression of one or a plurality of genes, selected from the group listed in Table 2:

03 BT / ALl 33

030303 BT / ALl 34

10. The method in accordance with one or a plurality off Claims 1 through 9, characterized in that one employs-- microorganisms of the corynebacterium glutamicum type.

11. Recombinant microorganisms, preferably coryneform bacteria, in which one or a plurality of the genes, selected from the yaeC, abc and yaeE group, which code for the uptake of methionine from the surrounding medium, coding for the MetD2 methionine uptake system, is or are present, attenuated, especially deactivated, or expressed at a lower level compared to the starting- organism without attenuated or deactivation of the MetD2 methionine uptake system.

12. A vector which contains a fragment with at least 15 sequenced nucleotides which contains a sequence or sequences of at least one of the yaeC, abc and yaeE genes and cannot be replicated into c. glutamicum.