Method for microbial production of amino acids of the spartate and/or glutamate family and agents which can be used in said method

Abstract

A method is disclosed for microbially producing L-amino acids of aspartate and/or glutamate families which comprises the step of: culturing a strain of Escherichia coli that produces said L-amino acids in a medium suitable for the production of an L-amino acid, whereby said Escherichia coli strain is transformed with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application Ser. No. 09/529,043, which is the US National Phase of PCT/EP98/06210 filed 30 Sep. 1998 and claiming the benefit of the priority of German Patent Application 197 43 894.6 filed 4 Oct. 1997 and German Patent Application 198 31 609.7 filed 14 Jul. 1998.

FIELD OF THE INVENTION

[0002] The invention relates to a method of microbial production of amino acids of the aspartate family and/or of the glutamate family, to the pyruvate-carboxylase gene and to gene structures, vectors and transformed cells containing the pyruvate carboxylate gene.

BACKGROUND OF THE INVENTION

[0003] Amino acids are of considerable economic interest since amino acids have many uses: thus, for example, L-lysine and L-threonine, L-methionine and L-tryptophan are necessary as fodder additives, L-glutamate as an additive to suppress L-isoleucine and L-tyrosine in the pharmaceutical industry, L-arginine and L-isoleucine as medicaments or L-glutamate, L-aspartate and L-phenylalanine as starting substances for the synthesis of fine chemicals.

[0004] A preferred method of producing these different amino acids is the biotechnical production by means of microorganisms such that in this manner the biologically-effective and optically-active forms of the respective amino acids are obtained and simple and inexpensive raw materials can be used. As microorganisms, for example, Corynebacterium glutamicum and its derivatives ssp. Flavum and ssp. Lactofermentum (Liebl et al., Int J System Bacteriol 1991, 41: 255 to 260) in addition to Escherichia coli and related bacteria are used. These bacteria normally produce the amino acids but only in amounts required for growth so that no surplus amino acids are formed and can be recovered. This is because in the cells the biosynthesis of -amino acids is controlled in many ways. As a consequence, there are already known various processes to increase the product formation by cutting out the control mechanisms. In these processes, for example, amino acid analogs are introduced to switch off the effective regulation of the biosynthesis. For example, a process has been used which is resistant to L-tyrosine analogs and L-phenylalanine analogs (JP 19037/1976 and 39517/1978). The processes also have been described in which bacteria resistant to L-lysine analogs or L-phenylalanine analogs have been used to suppress the control mechanisms (EP 0 205 849, GB 2 152 509).

[0005] Furthermore, microorganisms which have been constructed also by recombinant DNA-techniques obviate regulation of biosynthesis in that the gene which is coded in the no longer feedback-inhibited key.enzyme is cloned and expressed. For example, the recombinant L-lysine-producing bacterium with plasmid-coded feedback-resistant aspartate kinase is known (EP 0 381 527). In addition, a recombinant L-phenylalanine-producing bacterium with feedback-resistant prephenate dehydrogenase is, described (JP 123475/1986, EP 0 488 424).

[0006] In addition, by overexpression of genes which do not code for feedback-sensitive enzymes in amino acid synthesis, increased amino acid yields are obtainable. Thus, for example, lysine formation can be improved by increased synthesis of the dihydrodipicolinate synthesis (EP 0 197 335). Increasingly, by increased synthesis of the threoninedehydratease, improved isoleucine formation is achieved (EP 0 436 886).

[0007] Further investigations in increasing amino acid production have been targeted on the improved availability of the cellular primary metabolites of central metabolism. Thus it is known that, by recombinant techniques, over-expression of the transketolase can bring about an improved product formation of L-tryptophan or L-tyrosine or L-phenylalanine (EP 0 600 463). Furthermore, a reduction of the phosphoenolpyruvate-carboxylase activity in Corynebacterium leads to improved formation of aromatic amino acids (EP 0 3331 145) whereas by contrast an increase in the phosphoenolpyruvate-carboxylase activity in

[0008] Corynebacterium leads to increased recovery of amino acids of the aspartate family (EP 0 358 940).

[0009] During the growth and especially under amino acid production conditions, the tricarboxylic acid cycle must continuously and effectively be supplemented with C4 compounds, for example, oxalic acetate to replace intermediate products withdrawn for the amino acid biosynthesis. Until recently it has been thought that phosphoenolpyruvate-carboxylase was responsible for these so-called anaplerotic functions in Corynebacterium (Kinoshita, Biology of Industrial Micro-organisms 1985-115 to 142, Benjamin/Cummings Publishing Company, London; Liebl, The Prokaryotes II, 1991 to 1171, Springer Verlag N.Y.; Vallino and Stephanopoulos, Biotechnol Bioeng 1993, 41: 633 to 646).

[0010] It has, however, now been found that phosphoenolpyruvate-carboxylase-negative mutants grow equally by comparison to the-respective starting strains on all media. (Peters-Wendisch et al., FEMS Microbiology Letters 1993, 112: 269 to 274; Gubler et al., Appl Nicrobiol Biotechnol 1994, 40: 857 to 863). These results indicate that the phosphoenolpyruvate-carboxylase is not essential for the growth and plays no-role or only a small role for the anaplerotic reactions. Furthermore the aforementioned results indicate that in Corynebacterium another enzyme must be provided which is responsible for the synthesis of oxalacetate which is required for growth. Recently, indeed, a pyruvate-carboxylase activity has been found in permeablized cells of Corynebacterium glutamicum. (Peters-Wendisch et al., Microbiology 1997, 143: 1095 to 1103). This enzyme is effectively inhibited by AMP, ADP and acetyl coenzyme A and in the presence of lactate as a carbon source is formed in increased quantities. Since one must conclude that this enzyme is answerable primarily for the satisfaction of the tricarboxylic acid cycle of growth, it was to be expected that an increase in the gene expression or the enzymatic activity would either give rise to no increase in the amino acids belonging to the aspartate or yield only an increase therein. Furthermore, it was to be expected that an increase in the gene expression or the enzymatic activity of the pyruvate-carboxylase would also have no influence on the production of amino acids of other families.

SUMMARY OF THE INVENTION

[0011] It has surprisingly been found that an increase in the pyruvate-carboxylase activity by genetic modification of the enzyme and/or by increasing the pyruvate-carboxylase gene expression, the microbial production of amino acids of the aspartate and/or the glutamate families can be increased. It has been found that especially strains with increased copy numbers of the pyruvate-carboxylase gene can produce about 50% more lysine, 40% more threonine and 150% more homoserine in the culture medium. It has been found further that, surprisingly, the glutamate production is also significantly increased (compare especially the example under 6. Table 4).

[0012] The genetic alteration of the pyruvate-carboxylase to increase the enzyme activity is effected preferably by mutation of the endogenous gene. Such mutation can either be achieved by classical methods like, for example, by UV irradiation or by mutation triggering the chemicals or targeted by means of gene technological methods like deletion, insertion and/or nucleotide exchange.

[0013] The pyruvate-carboxylase gene expression is increased by increasing the gene copy number and/or by reinforcing regulatory factors which positively influence the expression of the gene. Thus a reinforcement of regulatory elements, preferably on the transcription plane can be effected in that especially the transcription signals are increased. This can be effected, for example, by varying the promoter sequence of the promoter preceding the structure gene to enhance its effectiveness or by replacing the promoter completely by more effective promoters. A reinforcement of the transcription can also be effected by a corresponding influence on a regulator gene associated with the pyruvate-carboxylase gene. This can be achieved, for example, by mutation of a regulatory gene sequence to influence the effectivity of the binding of a regulator protein to the DNA of the pyruvate-carboxylase gene which is regulated so that the transcription is thereby enhanced and thus the gene expression is increased. Furthermore the pyruvate-carboxylase gene can also be associated with a so-called "enhancer" as a regulatory sequence and which by means of an improved interchange between RNA polymerase and DNA also effects an increased pyruvate-carboxylase gene expression. However, a reinforcement of translations is also possible in that, for, example, the stability of the mRNA is improved.

[0014] For increasing the gene copy number the pyruvate-carboxylase gene is built into a gene construct or vector. The gene construct contains especially the regulatory sequences associated with the pyruvate-carboxylase gene, preferably those which reinforce the gene expression. For the incorporation of the pyruvate-carboxylase gene in a gene construct, the gene is progressively isolated from a microorganism strain of the Corynebacterium variety and is transformed in an amino-acid producing microorganism strain, especially Corynebacterium or in Escherichia coli or serratia marcenscens. For the process of the invention, especially genes from C. glutamicum or C. glutamicum ssp. flavum or C. glutamicum ssp. lactofermentum are suitable. After isolation of the gene and in the in vitro recombination with known vectors (see for example Simon et al., Bio/Technology 1983, 1: 784 to 791; Eikmanns et al., Gene 1991, 102: 93 to 98), .the transformation is effected in the amino-acid producing strain by electroporation (Liebl et al., FEMS Microbiology Letters 1991, 65: 299 to 304) or conjugation (Schafer et al., J. Baceriol 1990, 172: 1663 to 1666).

[0015] As the host strain preferably such amino acid producers are used which have been deregulated in the synthesis of the corresponding amino acid and/or show an increased export carrier activity for the corresponding amino acid. Furthermore, such strains are preferred which contain an increased number of such central metabolism metabolites as anticipated in the synthesis of, the corresponding amino-acid and/or strains which contain a reduced proportion of the central metabolism metabolites which do not participate in the synthesis of the corresponding amino acid, especially metabolites which tolerate competitive reactions; i.e. such strains are preferred in which synthesis paths competitive with the corresponding amino acid biosynthesis path run with reduced activity. Thus, especially a Coxyne-former microorganism strain with reduced citrate synthase activity is suitable as a strain resistant to L-agparaginic-acid-.beta.-methylester (AME) (EP 0 551 614).

[0016] After isolation, the pyruvate-carboxylase gene is obtained with nucleotide sequences which code for the amino acid sequence given under SEQ ID NO. 2 or their allele variations or the nucleotide sequence of nucleotides 165 to 3587 according to SEQ ID NO. 1 or a substantially identically-effective DNA sequence. The gene further contains a protein promoter of the: nucleotide sequence of nucleotides 20 to 109 according to SEQ ID NO. 1, a substantially identically effective DNA sequence. Allele variations or identically effective DNA sequences encompass especially functional derivations which are corresponding nucieotide sequences formed by deletions, insertions and/or substitutions of nucleotides whereby the enzyme activity or function remains or can even be increased. This pyruvate-carboxylase gene is preferably used in the process of the invention.

[0017] The pyruvate-carboxylase gene with or without the preceding promoter or with or without the associated regulator gene can be preceded by and/or followed by one or more DNA sequences so that the gene is contained in a gene structure.

[0018] The pyruvate-carboxylase gene is preferably preceded by the tac-promoter (lacI.sup.Q-Gen) which is associated especially with regulatory sequences.

[0019] By cloning the pyruvate-carboxylase gene, plasmids are obtained which contain the gene and are suitable for, transformation to an amino acid producer. The cells obtained by transformation which preferably correspond to transformed cells of Corynebacterium, contain the gene in replicatable form, i.e. in additional copies on the chromosome, whereby the gene copies are integrated by recombination at optional sites in the genome and/or on a plasmid or vector.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a physical map and restriction analysis of the cloned pyruvate carboxylase encoding gene from C. Glutamicum ATCC 13032 in pUC18 resulting in the vector pucpyc.

[0021] FIG. 2 is a physical map of the expression vector pUWEX1 containing the pyruvate carboxylase encoding gene from C. glutamicum 13032. Abbreviations: pvc=pyruvate carboxylase, Ptac-IPTG-inducibe synthetic promoter of trp-(-35 box) and lac-promoter (-10 box) regions; laclq-gene encoding the repressor of lac operon, kan-gene encoding the resistance to kananycin.

EXAMPLE

1. Cloning the Pyruvate-Carboxylase Gene of Corynebacterium Glutamicum

[0022] Starting from conserved regions of all prior known pyruvate-carboxylase-(pyc-) genes of Saccharomyces cerevisiae (J Biol Chem 1988, 263: 11493-11497; Mol Gen Genet 1991, 229: 307-315), Mensch (Biochem Biophys Acta 1994, 1227: 46-52):, Maus (Proc Natl Acad Sci, USA. 1993, 90: 1766-1770), Aedes aegypti (EMBL-GeneBank: Accession No. L36530) and from Mycobacterium tuberculosis (EMBL-GeneBank: Accession Nr. U00024). PCR primer is synthesized (MWG Biotech). The primer corresponds to the bases 810 to 831 and 1015 to 1037 of the pyc gene from M. tuberculosis. With this primer, by means of PCR according to the standard method of Innis et al (PCR protocols. A Guide to Methods and Applications, 1990, Academic Press) for nongenerated homologous primer, a fragment of about 200 bp of chromosomal DNA of C. glutamicum ATCC 13032 as-has been described by Eikmanns et al. (Microbiology 1994, 140: 1817-1828) is isolated following amplification. The size of 200 bp corresponds to the expectation for the pyc gene. The PCR product as described by Sanger et al (Proc Natl Acad Sci USA 1977, 74: 5463-5467) was sequenced. The sequencing was carried out with fluorescence-marked ddNTPs with an automatic DNA sequencing apparatus (Applied Biosystems).

[0023] Starting from this DNA fragment of C. glutamicum, the following homologous oligonucleotides are produced: TABLE-US-00001 pyc 1 5'- CGTCTTCATCGAAATGAAC-3' SEQ ID NO: 3 pyc 2 5'- ACGGTGGTGATCCGGCACT-3' SEQ ID NO: 4

[0024] The oligonucleotide is used as a PCR primer for isolating the probe for the gene of pyruvate-carboxylase (pyc) from C. glutamicum. The primer is introduced into a PCR reaction with chromosomal DNA from C. glutamicum and digoxygenine-marked nucleotides. The reaction is carried out in accordance with the instructions of the "PCR DIG Labeling Kits" of the firm Behringer Mannheim. With this approach, adigoxygenine-marked DNA fragment is amplified which corresponds to the expected size of about 200 bp. The thus produced pyc probe is then used to identify, utilizing. Southern-blot-hybridization, A DNA fragment in the chromosomal DNA of C. glutamicum on which the pyc gene is localized. For this purpose each 2 to 5 .mu.g of chromosomal DNA from C. glutamicum WT is cleaved with the restriction enzyme HindIII, SphI, SalI, DdraI, EcoRI and BamHI and the obtained DNA fragments are correspondingly separated by size over 16 hours at 20 volts gel-electrophoretically in an 0.8% agarose gel. The DNA fragments found in the agarose gel are denatured by the Southern blot (J Mol Biol 1975, 98: 503-517) and subjected to the vacuum-supported separation with the VacuGene Blot Apparatus of Pharmacia LKB (Uppsala, Sweden) from the gene matrix transferred onto a nylon membrane (Nytran N13 of Schleicher and Schull, Dassel, Switzerland), immobilized and the digoxygenine marker detected by means of NBT/X phosphate conversion with alkali phosphatizes in this manner. The following chromosomal fragments hybridized with the pyc-DNA-probe can be detected: a 17 kb HindIII-fragment, a 6.5 kb SalI fragment and a 1.35 kb EcoRI fragment.

[0025] The 17 kb HindIII fragment was isolated and subcloned. For this purpose a cosmid gene bank of chromosomal DNA from C. glutamicum in cosmid pH. C79 was used which represented the genome of C. glutainicum to 99% (Mol Microbiol 1992, 6: 317-326). The E. coli strain DH5.alpha. was transformed with this gene bank by means of the CaCl.sub.2 method of Sambrook et al (Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press) and plated out to about 300 colonies per LB-agar plate with 50 .mu.g/l kanamycin (a total of 5000 colonies). Then the obtained transformed product was transferred on a nytran N13 filter and incubated for 5 minutes for alkali lysis of the cells and denaturing of the DNA on Whatmann paper soaked with 0.5 M NaOH and 1.5 N NaCl. The subsequent neutralization is effected with 1 M Tris/HCl pH 7.5 and 1.5 M NaCl. The subsequent neutralization is effected with 1 M Tris/HCl pH 7.5 and 1.5 M NaCl.

[0026] After incubation of the filter in 2.times.SSC, the liberated DNA is fixed by UV radiation at 366 nm on the filter. Then the remaining cell fragments are removed by shaking in 3.times.SSC, 0.1% SDS at 50.degree. C. The filter in this form is used for the hybdridization with a specific pyc probe as described by Southern (J Mol Biol 1975, 98: 503-517).

[0027] The 3 transformands were identified from the pyc probe hybridization. From these transformands the cosmid DNA was isolated by means of plasmid proportion in accordance with the alkali lysis method of Birnboim (Meth Enzymol 1983, 100: 243-255) and then tested by restriction and Southern blot analysis for the presence of the HINDIII fragments. The cosmid pH C79-10 which contains a 40 kb HINDIII transmission completely and was further analyzed. It showed that also after the restriction with the endonucleosis SalI and EcoRI the same hybridized fragments as in the chromosomal DNA, i.e. a 6.5 kb SalI-fragment and a 1.35 kb EcRI-fragment. The 17 kb HindIII-fragment was isolated by restriction from the cosmid and is ligated in the E. coli vector pUC 18, which is also cleaved-with HindIII. A restriction analysis of the fragments in the resulting vector pUC pyc was carried out. The physical mapping of the fragments is shown in FIG. 1.

2. Sequencing of the Pyruvate-Carboxylase Gene

[0028] In further subcloning steps a 0.85 kb SalI-EcoRI-fragment was isolated from the plasmid pUC pyc by restriction with corresponding restriction enzymes as a 1.35 kb EcoRI-fragment, a 1.6 kb EcoRI-EcoRI-StuI-fragment as well as a 1.6 kb ClaI-fragment, that overlapped with 0.85 kb SalI-EcoRI-fragment. By ligation the fragments were cloned correspondingly in the restricting vector pUC 18 and then sequenced as described-above according to Sanger et al. In (Proc Natl Acad Sci USA 1977, 74: 5463-5467) the nucleotide sequences obtained were analyzed. The program package HUSAR (Release 3.0) of the German zone for cancer research (Heidelberg). The sequence analysis of the fragments gave a continuously open reading raster of 3576 bp which coded for a protein sequence of 1140 amino acids. Comparison of the protein sequence with the EMBL gene data bank (Heidelberg) gave similarities to all known pyruvate carboxylases. The highest identity (62%) was to the putative pyruvate-carboxylase from Mycobacterium tuberculosis (EMBL-GeneBank: Accession No. U00024). The similarity amounted to 76% when conserved amino acid exchange was followed. A comparison with the pyruvate-carboxylase of other organisms yielded an identity of 46 to 47% identical and 64 to 65% similar amino acids (Gene 1997, 191: 47-50; J Bacteriol 19.96, 178: 5960-5970; Proc Natl Acad Sci USA 1993, 990: 176.6-1770; Biochem J.1996, 316:. 631-637; EMBL-GenBank: Accession No. L36530; J Biol Chem 1988, 263: 11493-11497; Mol Gen Genet 1991, 229: 307-315). From these results it could be concluded that the cloned fraction base was the gene for the pyruvate-carboxylase from C. glutamicum. The nucleotide sequence of the gene is given under SEQ ID NO: 1 and the corresponding amino acid sequence under SEQ ID NO: 2.

3. Overexpression of the Pyruvate-Carboxylase

[0029] For the overexpression of the gene for pyruvate-carboxylase from C. glutamicum, the gene was cloned from the plasmid pUCpyc as the 6.2 kb Sspl-Scal-fragment in the E. coli glutamicum swing vector PEKO (Gene 1991, 102: 93-98) which was cleaved with the restriction endonucleosis EcbRI and PstI. By means of Klenow-polymerase treatment the overhanging ends were ligated to smooth ends by filling the EcoRI or linking PstI and the linearized vector was ligated with the 6.2 kb Sspl-Scal-fragment. The resulting construct pEK0pyc was additionally. transformed in the E. coli strain DH5.alpha., the plasmid DNA was isolated on the resulting transformand and the correctness of the inserts controlled by restriction. The DNA was then introduced in the strain SP 733 by electroporation (FEMS Nicrobiol Lett 1989, 65: 299-304).

[0030] This strain is a mutant of the restriction negative C. glutamicum strain R 127 (Dechema Biotechnology Conference 1990, 4: 323-327, Verlag Chemie) which was obtained by chemical mutagenesis and was characterized in that it cannot be grown on a minimal medium with pyruvate and lactate as single carbon sources (Microbiology 1997, 143: 1095-1103). This phenotype is recognized as a defect in the pyruvate-carboxylase and can be complemented by introducing the pyruvate-carboxylase gene from C. glutamicum, i.e. the strain which is carried by the plasmid-pEK0pyc and was by contrast to the starting strain able to grow again in the presence of minimal medium with lactate as a single carbon source. This was a verification that the gene was coded for a functional pyruvate-carboxylase.

[0031] Furthermore, the plasmid pEK0pyc was transformed in the C. glutamicum wild type ATCC 13032 by electroporation. The resulting strain WT (pEK0pyc) was investigated by comparison to the wild type ATCC 13032 with respect to its pyruvate-carboxylase activity. The strain was cultured in a complex medium (Luria-Bertani, Molecular Cloning, A laboratory manual, 1989, Cold Spring Harbor Laboratory Press) with 0.5% lactate and on minimal medium with 2% lactate or 4% glucose and the pyruvate-carboxylase test was-carried out corresponding to the method as described by Peters-Wendisch et al (Microbiology 1997, 143: 1095-1103). The results of the analysis (Table 1) showed that the pyruvate-carboxylase activity in the pEK0-pyc-carrying strain was about 4 times higher than in the starting strain.

4. Increased Accumulation of Lysine by Overexpression of the Pyruvate-Carboxylase Gene in the Strain C-glutamicum DG 52-5.

[0032] To investigate the effect of the overexpression of the gene for the pyruvate-carboxylase in the lysine-producing strain DG 52-5. (J Gen Microbiol 1988,134: 3221-3229), the expression vector pVWEX1 is used to promote an IPTG-inducible expression. In this vector, the pyc gene was promotorlessly cloned. For that purpose, initially PCT-Primer (Primer 1=Position 112-133; Primer 2-=Position 373 to 355 in the nucleotide sequence according to SEQ ID-NO. 1), is synthesized and 261 bp of the promotorless starting region of the pyruvate-carboxylase gene was amplified by means of PCR. The primer was so selected that Primer I enabled a PstI cleavage site and Primer 2 a BamHI cleavage site. After the PCR, the 274 bp PCR product was isolated, ligated to concatemers and then cleaved with the restriction enzymes PstI and BamHI. The restriction product was concentrated by ethanol precipitation and then ligated with the PstI-BamHI cleaved vector pVWEX1. The resulting construct pVWEX1-PCR was tested by restriction. The end region of the pyc gene was isolated by RcaI-Klenow-SalI treatment from the vector pEK0pyc and ligated in the BamHI-Klenow-SalI during vector PVWEX1-PCR. The resulting construct pVWEX1pyc was analyzed by restriction mapping. Physical mapping of the plasmid is shown in FIG. 2.

[0033] The plasmid was introduced by electroporation in the C. glutamicum strain DG 52-5. As a control, the strain DG 52-5 was transformed with the vector pVWEX1 without insert-and the L-lysine precipitation of three different transformands was compared. For this purpose (DG 52-5 (pVWEX1pyc) 3,4 and (2.times.TY; Molecular Cloning, A laboratory manual, 1989, Cold Spring Harbor Laboratory Press with 50 .mu.g/I kanamycin) and the respective fermentation medium in each case from the preculture was separately inoculated. The medium contained additional kanamycin to maintain the plasmid stable. In each case two parallel tests were run whereby one flask of 200 .mu.g IPTG/ml was added while the second flask contained no IPTG. After cultivation for 48 hours, at 30.degree. C. on a rotation shaker at 120 RPM, the accumulated lysine quantity in the medium was determined. The determination of the amino acid concentration was effected by means of high-pressure liquid chromatography (J. Chromat 1983, 266; 471-482).

[0034] The results of the fermentation are shown in Table 2 whereby the values given are mean values each from three experiments with different clones. It shows that the overexpression of the pyruvate-carboxylase gene results in a 50% increased accumulation of lysine in the medium. Thus the use of the covered and described gene for the anaplerotic enzyme-pyruvate-carboxylase enables a process of lysine formation to be significantly improved.

5. Increased Accumulation of Threonine and Homoserine by Overexpression of the Pyruvate-Carboxylase Gene in the Strain C. glutamicum DM 368-3

[0035] Analogously to the experiment in L-lysine formation, the accumulation of threonine in the culture supernatant by overexpression of the gene for pyruvate-carboxylase was also investigated for this purpose, as has been described under point 4, the threonine production strain C. glutamicum DM 368-3 (Degussa AG) was transformed with the plasmid pVWEX1pyc with control by the plasmid pVWEX1 and the threonine separation was, investigated with each of three different transformands. For this purpose DM 368-3 (pVWEX1) 2 and 3 and DM 368-3 (pVWEX1pyc) 1, 2 and 3 in complex medium (2.times.TY with 50 .mu.g/l kanamycin) were cultured and the fermentation medium CGXII (J Bacteriol 1993, 175: 5595-5603) in each case was separately inoculated from the preculture. The medium contained additional kanamycin to hold the plasmid stable. Two parallel sets of tests were carried out whereby 200 .mu.g IPTG/ml was added to one flask while the second flask contained no IPTG. After culturing for 48 hours at 30.degree. C. on a rotation shaker at 120 RPM, the threonine quantities accumulated in the medium were determined. The determination of the amino acid concentration was effected also by means of high-pressure liquid chromatography (J Chromat 1983, 266: 471-482). The results of the fermentation are shown in Table 3 whereby the values given are mean values from each of three experiments with different clones. It shows that the overexpression of the pyruvate-carboxylase gene gave about a 40% increase in the threonine concentration in the medium. The use of the covered and described gene for anaplerotic enzyme pyruvate-carboxylase in a process for L-threonine formation significantly improves the latter.

[0036] Furthermore, the amino acid concentration determination shows surprisingly that the strain with the overexpressed pyruvate-carboxylase gene also yields 150% more homoserine in the medium than the strain with the nonoverexpressed gene. Corresponding results are shown in Table 3. They make clear that in the process according to the invention the threonine yield like the homoserine yield can be significantly improved.

6. Increased Accumulation of Glutamate by Overexpression of the Pyruvate-Carboxylase Gene in C. glutamicum Wild Type

[0037] Analogous to the experiments for L-lysine, L-threonine and L-homoserine formation (see above, the 4. and 5.), accumulation of glutamate in the culture supernatant, overexpression of the gene for pyruvate-carboxylase was also investigated. For this purpose, as described, the point 4 wild type C-glutamicum ATCC 13032 with the plasmid pVWEX1 pyc was transformed in addition to the control with the plasmid pVWEX1 and the glutamate separation determined from each of two different transformands. Thus C. glutamicum ATCC 13032 pVWEX1pyc) D1 and D2 as well as C. glutamicum ATCC 13032,(pVWEX1 pyc) 1 and 2 were cultured in the complex medium (2.times.TY with 50 .mu.g/l kanamycin) and the fermentation medium CGXII (J Bacteriol 1993, 175: 5595-5603) in each case was separately inoculated from the preculture period. The medium contained additional kanamycin to stabilize the plasmid. To induce glutamate separation, 25 mg Tween 60 was added per ml to the medium about 6 hours after the inoculation. Two parallel sets of tests were carried out whereby in one, 200 .mu.g IPTG/ml is added to the flask while the second flask contained no IPTG. After culturing for 48 hours at 30.degree. C. on a rotation shaker at 120 RPM, the glutamate quantity accumulated in the medium was determined. The determination of the amino acid concentration was effected also by means of high-pressure liquid chromatography (J Chromat 1983, 266; 471-482). The results of the fermentation are shown in Table 4 whereby values given are averages with each two experiments with different clones. It shows that the overexpression of the pyruvate-carboxylase gene gave rise to up to 500% increase of the glutamate concentration in the medium. The use of the covered and described gene for the anaplerotic enzyme pyruvate-carboxylase improved the glutamate formation significantly.

Sequence CWU 1

1

4 1 3728 DNA Corynebacterium glutamicum CDS (165)..(3587) pyruvate carboxylase 1 cgcaaccgtg cttgaagtcg tgcaggtcag gggagtgttg cccgaaaaca ttgagaggaa 60 aacaaaaacc gatgtttgat tgggggaatc gggggttacg atactaggac gcagtgactg 120 ctatcaccct tggcggtctc ttgttgaaag gaataattac tcta gtg tcg act cac 176 Val Ser Thr His 1 aca tct tca acg ctt cca gca ttc aaa aag atc ttg gta gca aac cgc 224 Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu Val Ala Asn Arg 5 10 15 20 ggc gaa atc gcg gtc cgt gct ttc cgt gca gca ctc gaa acc ggt gca 272 Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu Glu Thr Gly Ala 25 30 35 gcc acg gta gct att tac ccc cgt gaa gat cgg gga tca ttc cac cgc 320 Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly Ser Phe His Arg 40 45 50 tct ttt gct tct gaa gct gtc cgc att ggt acc gaa ggc tca cca gtc 368 Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu Gly Ser Pro Val 55 60 65 aag gcg tac ctg gac atc gat gaa att atc ggt gca gct aaa aaa gtt 416 Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala Ala Lys Lys Val 70 75 80 aaa gca gat gcc att tac ccg gga tac ggc ttc ctg tct gaa aat gcc 464 Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu Ser Glu Asn Ala 85 90 95 100 cag ctt gcc cgc gag tgt gcg gaa aac ggc att act ttt att ggc cca 512 Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr Phe Ile Gly Pro 105 110 115 acc cca gag gtt ctt gat ctc acc ggt gat aag tct cgc gcg gta acc 560 Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser Arg Ala Val Thr 120 125 130 gcc gcg aag aag gct ggt ctg cca gtt ttg gcg gaa tcc acc ccg agc 608 Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu Ser Thr Pro Ser 135 140 145 aaa aac atc gat gag atc gtt aaa agc gct gaa ggc cag act tac ccc 656 Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly Gln Thr Tyr Pro 150 155 160 atc ttt gtg aag gca gtt gcc ggt ggt ggc gga cgc ggt atg cgt ttt 704 Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg Gly Met Arg Phe 165 170 175 180 gtt gct tca cct gat gag ctt cgc aaa tta gca aca gaa gca tct cgt 752 Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr Glu Ala Ser Arg 185 190 195 gaa gct gaa gcg gct ttc ggc gat ggc gcg gta tat gtc gaa cgt gct 800 Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr Val Glu Arg Ala 200 205 210 gtg att aac cct cag cat att gaa gtg cag atc ctt ggc gat cac act 848 Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu Gly Asp His Thr 215 220 225 gga gaa gtt gta cac ctt tat gaa cgt gac tgc tca ctg cag cgt cgt 896 Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser Leu Gln Arg Arg 230 235 240 cac caa aaa gtt gtc gaa att gcg cca gca cag cat ttg gat cca gaa 944 His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His Leu Asp Pro Glu 245 250 255 260 ctg cgt gat cgc att tgt gcg gat gca gta aag ttc tgc cgc tcc att 992 Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe Cys Arg Ser Ile 265 270 275 ggt tac cag ggc gcg gga acc gtg gaa ttc ttg gtc gat gaa aag ggc 1040 Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val Asp Glu Lys Gly 280 285 290 aac cac gtc ttc atc gaa atg aac cca cgt atc cag gtt gag cac acc 1088 Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln Val Glu His Thr 295 300 305 gtg act gaa gaa gtc acc gag gtg gac ctg gtg aag gcg cag atg cgc 1136 Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys Ala Gln Met Arg 310 315 320 ttg gct gct ggt gca acc ttg aag gaa ttg ggt ctg acc caa gat aag 1184 Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu Thr Gln Asp Lys 325 330 335 340 atc aag acc cac ggt gca gca ctg cag tgc cgc atc acc acg gaa gat 1232 Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile Thr Thr Glu Asp 345 350 355 cca aac aac ggc ttc cgc cca gat acc gga act atc acc gcg tac cgc 1280 Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile Thr Ala Tyr Arg 360 365 370 tca cca ggc gga gct ggc gtt cgt ctt gac ggt gca gct cag ctc ggt 1328 Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala Ala Gln Leu Gly 375 380 385 ggc gaa atc acc gca cac ttt gac tcc atg ctg gtg aaa atg acc tgc 1376 Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val Lys Met Thr Cys 390 395 400 cgt ggt tcc gac ttt gaa act gct gtt gct cgt gca cag cgc gcg ttg 1424 Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala Gln Arg Ala Leu 405 410 415 420 gct gag ttc acc gtg tct ggt gtt gca acc aac att ggt ttc ttg cgt 1472 Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile Gly Phe Leu Arg 425 430 435 gcg ttg ctg cgg gaa gag gac ttc act tcc aag cgc atc gcc acc gga 1520 Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg Ile Ala Thr Gly 440 445 450 ttc att gcc gat cac ccg cac ctc ctt cag gct cca cct gct gat gat 1568 Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro Pro Ala Asp Asp 455 460 465 gag cag gga cgc atc ctg gat tac ttg gca gat gtc acc gtg aac aag 1616 Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val Thr Val Asn Lys 470 475 480 cct cat ggt gtg cgt cca aag gat gtt gca gct cct atc gat aag ctg 1664 Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro Ile Asp Lys Leu 485 490 495 500 cct aac atc aag gat ctg cca ctg cca cgc ggt tcc cgt gac cgc ctg 1712 Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser Arg Asp Arg Leu 505 510 515 aag cag ctt ggc cca gcc gcg ttt gct cgt gat ctc cgt gag cag gac 1760 Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu Arg Glu Gln Asp 520 525 530 gca ctg gca gtt act gat acc acc ttc cgc gat gca cac cag tct ttg 1808 Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala His Gln Ser Leu 535 540 545 ctt gcg acc cga gtc cgc tca ttc gca ctg aag cct gcg gca gag gcc 1856 Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro Ala Ala Glu Ala 550 555 560 gtc gca aag ctg act cct gag ctt ttg tcc gtg gag gcc tgg ggc ggc 1904 Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu Ala Trp Gly Gly 565 570 575 580 gcg acc tac gat gtg gcg atg cgt ttc ctc ttt gag gat ccg tgg gac 1952 Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu Asp Pro Trp Asp 585 590 595 agg ctc gac gag ctg cgc gag gcg atg ccg aat gta aac att cag atg 2000 Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val Asn Ile Gln Met 600 605 610 ctg ctt cgc ggc cgc aac acc gtg gga tac acc ccg tac cca gac tcc 2048 Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro Tyr Pro Asp Ser 615 620 625 gtc tgc cgc gcg ttt gtt aag gaa gct gcc agc tcc ggc gtg gac atc 2096 Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser Gly Val Asp Ile 630 635 640 ttc cgc atc ttc gac gcg ctt aac gac gtc tcc cag atg cgt cca gca 2144 Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln Met Arg Pro Ala 645 650 655 660 atc gac gca gtc ctg gag acc aac acc gcg gta gcc gag gtg gct atg 2192 Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala Glu Val Ala Met 665 670 675 gct tat tct ggt gat ctc tct gat cca aat gaa aag ctc tac acc ctg 2240 Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys Leu Tyr Thr Leu 680 685 690 gat tac tac cta aag atg gca gag gag atc gtc aag tct ggc gct cac 2288 Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys Ser Gly Ala His 695 700 705 atc ttg gcc att aag gat atg gct ggt ctg ctt cgc cca gct gcg gta 2336 Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg Pro Ala Ala Val 710 715 720 acc aag ctg gtc acc gca ctg cgc cgt gaa ttc gat ctg cca gtg cac 2384 Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp Leu Pro Val His 725 730 735 740 gtg cac acc cac gac act gcg ggt ggc cag ctg gca acc tac ttt gct 2432 Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala Thr Tyr Phe Ala 745 750 755 gca gct caa gct ggt gca gat gct gtt gac ggt gct tcc gca cca ctg 2480 Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala Ser Ala Pro Leu 760 765 770 tct ggc acc acc tcc cag cca tcc ctg tct gcc att gtt gct gca ttc 2528 Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile Val Ala Ala Phe 775 780 785 gcg cac acc cgt cgc gat acc ggt ttg agc ctc gag gct gtt tct gac 2576 Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu Ala Val Ser Asp 790 795 800 ctc gag ccg tac tgg gaa gca gtg cgc gga ctg tac ctg cca ttt gag 2624 Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr Leu Pro Phe Glu 805 810 815 820 tct gga acc cca ggc cca acc ggt cgc gtc tac cgc cac gaa atc cca 2672 Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg His Glu Ile Pro 825 830 835 ggc gga cag ttg tcc aac ctg cgt gca cag gcc acc gca ctg ggc ctt 2720 Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr Ala Leu Gly Leu 840 845 850 gcg gat cgt ttc gaa ctc atc gaa gac aac tac gca gcc gtt aat gag 2768 Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala Ala Val Asn Glu 855 860 865 atg ctg gga cgc cca acc aag gtc acc cca tcc tcc aag gtt gtt ggc 2816 Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser Lys Val Val Gly 870 875 880 gac ctc gca ctc cac ctc gtt ggt gcg ggt gtg gat cca gca gac ttt 2864 Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp Pro Ala Asp Phe 885 890 895 900 gct gcc gat cca caa aag tac gac atc cca gac tct gtc atc gcg ttc 2912 Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser Val Ile Ala Phe 905 910 915 ctg cgc ggc gag ctt ggt aac cct cca ggt ggc tgg cca gag cca ctg 2960 Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp Pro Glu Pro Leu 920 925 930 cgc acc cgc gca ctg gaa ggc cgc tcc gaa ggc aag gca cct ctg acg 3008 Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys Ala Pro Leu Thr 935 940 945 gaa gtt cct gag gaa gag cag gcg cac ctc gac gct gat gat tcc aag 3056 Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala Asp Asp Ser Lys 950 955 960 gaa cgt cgc aat agc ctc aac cgc ctg ctg ttc ccg aag cca acc gaa 3104 Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro Lys Pro Thr Glu 965 970 975 980 gag ttc ctc gag cac cgt cgc cgc ttc ggc aac acc tct gcg ctg gat 3152 Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr Ser Ala Leu Asp 985 990 995 gat cgt gaa ttc ttc tac ggc ctg gtc gaa ggc cgc gag act ttg atc 3200 Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg Glu Thr Leu Ile 1000 1005 1010 cgc ctg cca gat gtg cgc acc cca ctg ctt gtt cgc ctg gat gcg atc 3248 Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val Arg Leu Asp Ala Ile 1015 1020 1025 tct gag cca gac gat aag ggt atg cgc aat gtt gtg gcc aac gtc aac 3296 Ser Glu Pro Asp Asp Lys Gly Met Arg Asn Val Val Ala Asn Val Asn 1030 1035 1040 ggc cag atc cgc cca atg cgt gtg cgt gac cgc tcc gtt gag tct gtc 3344 Gly Gln Ile Arg Pro Met Arg Val Arg Asp Arg Ser Val Glu Ser Val 1045 1050 1055 1060 acc gca acc gca gaa aag gca gat tcc tcc aac aag ggc cat gtt gct 3392 Thr Ala Thr Ala Glu Lys Ala Asp Ser Ser Asn Lys Gly His Val Ala 1065 1070 1075 gca cca ttc gct ggt gtt gtc acc gtg act gtt gct gaa ggt gat gag 3440 Ala Pro Phe Ala Gly Val Val Thr Val Thr Val Ala Glu Gly Asp Glu 1080 1085 1090 gtc aag gct gga gat gca gtc gca atc atc gag gct atg aag atg gaa 3488 Val Lys Ala Gly Asp Ala Val Ala Ile Ile Glu Ala Met Lys Met Glu 1095 1100 1105 gca aca atc act gct tct gtt gac ggc aaa atc gat cgc gtt gtg gtt 3536 Ala Thr Ile Thr Ala Ser Val Asp Gly Lys Ile Asp Arg Val Val Val 1110 1115 1120 cct gct gca acg aag gtg gaa ggt ggc gac ttg atc gtc gtc gtt tcc 3584 Pro Ala Ala Thr Lys Val Glu Gly Gly Asp Leu Ile Val Val Val Ser 1125 1130 1135 1140 taa acctttctgt aaaaagcccc gcgtcttcct catggaggag gcggggcttt 3637 ttgggccaag atgggagatg ggtgagttgg atttggtctg attcgacact tttaagggca 3697 gagatttgaa gatggagacc aaggctcaaa g 3728 2 1140 PRT Corynebacterium glutamicum 2 Val Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu 1 5 10 15 Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu 20 25 30 Glu Thr Gly Ala Ala Thr Val Ala Ile Tyr Pro Arg Glu Asp Arg Gly 35 40 45 Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu 50 55 60 Gly Ser Pro Val Lys Ala Tyr Leu Asp Ile Asp Glu Ile Ile Gly Ala 65 70 75 80 Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu 85 90 95 Ser Glu Asn Ala Gln Leu Ala Arg Glu Cys Ala Glu Asn Gly Ile Thr 100 105 110 Phe Ile Gly Pro Thr Pro Glu Val Leu Asp Leu Thr Gly Asp Lys Ser 115 120 125 Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu 130 135 140 Ser Thr Pro Ser Lys Asn Ile Asp Glu Ile Val Lys Ser Ala Glu Gly 145 150 155 160 Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg 165 170 175 Gly Met Arg Phe Val Ala Ser Pro Asp Glu Leu Arg Lys Leu Ala Thr 180 185 190 Glu Ala Ser Arg Glu Ala Glu Ala Ala Phe Gly Asp Gly Ala Val Tyr 195 200 205 Val Glu Arg Ala Val Ile Asn Pro Gln His Ile Glu Val Gln Ile Leu 210 215 220 Gly Asp His Thr Gly Glu Val Val His Leu Tyr Glu Arg Asp Cys Ser 225 230 235 240 Leu Gln Arg Arg His Gln Lys Val Val Glu Ile Ala Pro Ala Gln His 245 250 255 Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe 260 265 270 Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val 275 280 285 Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln 290 295 300 Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys 305 310 315 320 Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu 325 330 335 Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile 340 345 350 Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile 355 360 365 Thr Ala Tyr Arg Ser Pro Gly Gly Ala Gly Val Arg Leu Asp Gly Ala 370 375 380 Ala Gln Leu Gly Gly Glu Ile Thr Ala His Phe Asp Ser Met Leu Val 385 390 395 400 Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala 405 410 415 Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile 420 425 430 Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg 435 440 445 Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro 450 455 460 Pro Ala Asp Asp Glu Gln Gly Arg Ile Leu Asp Tyr Leu Ala Asp Val 465 470 475 480 Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro 485 490 495 Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser 500 505 510 Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu 515 520 525 Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala 530

535 540 His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro 545 550 555 560 Ala Ala Glu Ala Val Ala Lys Leu Thr Pro Glu Leu Leu Ser Val Glu 565 570 575 Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu 580 585 590 Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val 595 600 605 Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro 610 615 620 Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser 625 630 635 640 Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln 645 650 655 Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala 660 665 670 Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys 675 680 685 Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys 690 695 700 Ser Gly Ala His Ile Leu Ala Ile Lys Asp Met Ala Gly Leu Leu Arg 705 710 715 720 Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp 725 730 735 Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala 740 745 750 Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala 755 760 765 Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile 770 775 780 Val Ala Ala Phe Ala His Thr Arg Arg Asp Thr Gly Leu Ser Leu Glu 785 790 795 800 Ala Val Ser Asp Leu Glu Pro Tyr Trp Glu Ala Val Arg Gly Leu Tyr 805 810 815 Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg 820 825 830 His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr 835 840 845 Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala 850 855 860 Ala Val Asn Glu Met Leu Gly Arg Pro Thr Lys Val Thr Pro Ser Ser 865 870 875 880 Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp 885 890 895 Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser 900 905 910 Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp 915 920 925 Pro Glu Pro Leu Arg Thr Arg Ala Leu Glu Gly Arg Ser Glu Gly Lys 930 935 940 Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala 945 950 955 960 Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro 965 970 975 Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr 980 985 990 Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg 995 1000 1005 Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val Arg 1010 1015 1020 Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn Val Val 1025 1030 1035 1040 Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg Asp Arg Ser 1045 1050 1055 Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp Ser Ser Asn Lys 1060 1065 1070 Gly His Val Ala Ala Pro Phe Ala Gly Val Val Thr Val Thr Val Ala 1075 1080 1085 Glu Gly Asp Glu Val Lys Ala Gly Asp Ala Val Ala Ile Ile Glu Ala 1090 1095 1100 Met Lys Met Glu Ala Thr Ile Thr Ala Ser Val Asp Gly Lys Ile Asp 1105 1110 1115 1120 Arg Val Val Val Pro Ala Ala Thr Lys Val Glu Gly Gly Asp Leu Ile 1125 1130 1135 Val Val Val Ser 1140 3 19 DNA Artificial Sequence Description of Artificial Sequence PCR primer 3 cgtcttcatc gaaatgaac 19 4 19 DNA Artificial Sequence Description of Artificial Sequence PCR primer 4 acggtggtga tccggcact 19

Claims

1. A method of microbially producing L-amino acids of aspartate and/or glutamate families which comprises the step of: culturing a strain of Escherichia coli that produces said L-amino acids in a medium suitable for the production of an L-amino acid whereby said Escherichia coli strain is transformed with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.

2. A method of microbially producing L-amino acids of aspartate and/or glutamate families which comprises the step of: culturing a strain of Escherichia coli that produces said L-amino acids in a medium suitable for the production of an L-amino acid, whereby said Escherichia coli strain comprises an isolated polynucleotide, whereby said polynucledtide encoding an enzyme with pyruvate carboxylase activity comprises the amino acid sequence having SEQ ID NO: 2.

3. The method according to claim 2 wherein said polynucleotide encoding an enzyme with pyruvate carboxylase activity comprises SEQ ID NO:1 or a nucleotide sequence with positions:165 to 3587 of SEQ ID NO:1.

4. The method according to claim 2 wherein said polynucleotide is isolated from a microorganism of the genus Corynebacterium.

5. The method according to claim 2 wherein a strain of Escherichia coli is transformed with an isolated polynucleotide which codes for an enzyme with pyruvate carboxylase activity comprising the amino acid sequence of SEQ ID NO:2.

6. The method according to claim 3 wherein a strain of Escherichia coli is transformed with an isolated polynucleotide which codes for an enzyme with pyruvate carboxylase activity comprising the amino acid sequence of SEQ ID NO:2.

7. The method according to claim 2 whereby the amino acid of the aspartate and/or glutamate family produced is selected from the group consisting of L-lysine, L-threonine, L-arginine, and glutamate.

8. A method of microbially producing L-amino acids of aspartate and/or glutamate family, which comprises the step of: transforming a strain of Escherichia coli with an isolated polynucleotide which codes for an enzyme with pyruvate carboxylase activity comprising a polynucleotide having the amino acid sequence of SEQ ID NO:2; and culturing said transformed strain in a medium suitable for the production of an L-amino acid and-producing said L-amino acids.

9. The method according to claim 8 whereby the L-amino acid of the aspartate and/or glutamate family produced is selected from the group consisting of L-lysine, L-threonine, L-arginine, and glutamate.

10. A method for microbially overproducing L-threonine in a culture medium, which comprises the step of: (a) providing a strain of Escherichia coli transformed with an isolated polynucleotide encoding a pyruvate carboxylase polypeptide comprising the amino acid sequence of SEQ ID NO:2 operatively linked to a promoter and capable of producing threonine; (b) culturing the transformed strain of Escherichia coli under conditions which permit expression of the pyruvate carboxylase polypeptide to cause the overproduction of threonine compared to an untransformed form of the strain of Escherichia coli; and (c) isolating the threonine produced by the transformed strain of Escherichia coli.

11. The method for microbially overproducing L-threonine defined in claim 10 wherein according to step (b) the expression by the isolated polynucleotide of the pyruvate carboxylase polypeptide is increased by increasing a copy number of the isolated polynucleotide.

12. The method for microbially overproducing L-threonine defined in claim 11, wherein increasing the copy number of the isolated polynucleotide is achieved by transforming said Escherichia coli with a vector comprising the isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2 in association with a regulatory sequence.

13. The method for microbially overproducing L-threonine defined in claim 12, wherein said isolated polynucleotide comprises the nucleotide sequence of nucleotides 165 to 3587 of SEQ ID NO:1.

14. The method for microbially overproducing L-threonine defined in claim 13, wherein said isolated polynucleotide comprises the nucleotide sequence of SEQ ID NO:1.

15. The method for microbially overproducing L-threonine defined in claim 10 wherein according to step (b) the pyruvate carboxylase having SEQ ID NO:2 participating in the synthesis of L-threonine is deregulated and/or wherein an enhanced expert carrier activity is shown for the threonine.

16. The method for microbially overproducing L-threonine defined in claim 10 wherein according to step (a) the transformed Escherichia coli has a high proportion of central metabolism metabolites of the L-threonine participating in the synthesis.

17. The method for microbially overproducing L-threonine defined in claim 10 wherein according to step (a) the transformed Escherichia coli has biosynthesis paths which run with reduced activity when competing with biosynthesis paths of L-threonine.

18. The method for microbially overproducing L-threonine defined in claim 17 wherein according to step (a) the isolated polynucleotide encoding a pyruvate carboxylase polypeptide comprising the amino acid sequence of SEQ-ID NO:2 is isolated from a microorganism strain of the variety Corynebacterium.

19. The method for microbially overproducing L-threonine defined in claim 10 wherein according to step (a) the expression of the polynucleotide encoding the polypeptide having SEQ ID NO:2 is increased by reinforcement of the transcription signal.

20. The method for microbially overproducing L-threonine defined in claim 10 wherein according to step (a) the promoter linked to the polynucleotide encoding the polypeptide of SEQ ID NO:2 is the tac promoter located ahead of the polynucleotide.

21. The method for microbially overproducing L-threonine defined in claim 20 wherein the tac promoter is associated with regulatory-sequences.

22. The method for microbially overproducing L-threonine defined in claim 10 wherein the transformed Escherichia coli contains a vector pVWEX1pyc.

23. The method for microbially overproducing L-threonine defined in claim 12 wherein the vector comprising the isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is pVWEX1pyc.

24. An Escherichia coli cell that produces L-amino acids of the aspartate and/or glutamate families, transformed with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.

25. The Escherichia coli cell defined in claim 24 transformed with an isolated polynucleotide encoding an enzyme with pyruvate carboxylase activity comprising the amino acid sequence having SEQ ID NO:2.

26. The Escherichia coli cell defined in claim 24 transformed with the vector pVWEX1pyc.

27. A method of making a transformed Escherichia coli cell that produces L-amino acids of the aspartate and/or glutamate families, which comprises the step of transforming the Escherichia coli cell with a gene construct comprising a polynucleotide encoding an enzyme with pyruvate carboxylase activity.

28. The method of making a transformed Escherichia coli cell defined in claim 26 wherein the polynucleotide is an isolated polynucleotide encoding an enzyme with pyruvate carboxylase activity comprising the amino acid sequence of SEQ ID NO: 2

29. The method of making a transformed Escherichia coli cell defined in claim 26 wherein the Escherichia coli cell is transformed with the vector pVWEX1pyc.