KR100526316B1 - Method for microbial production of amino acids of the aspartate and/or glutamate family and agents which can be used in said method - Google Patents

Abstract

      The present invention relates to a method for microbial preparation of aspartate and / or glutamate amino acids, wherein the pyruvate carboxylase activity is the genetic of the enzyme and / or pyruvate carboxylase gene expression of the microorganism that will produce the corresponding amino acid. Increases with change. The present invention also relates to pyruvate carboxylase genes and agents which can be used in the above methods.

Description

METHOD FOR MICROBIAL PRODUCTION OF AMINO ACIDS OF THE ASPARTATE AND / OR GLUTAMATE FAMILY AND AGENTS WHICH CAN BE USED IN SAID METHOD}

The present invention provides a method for producing microorganisms of aspartate and glutamate-type amino acids shown in claims 1 to 17, pyruvate-carboxylase genes shown in 18 to 23, gene structure shown in claim 24, vector shown in claim 25, claim 26 To transformed cells as shown in Figures 31 to 31, their use as set forth in claims 32 to 37.

Amino acids have economic value because of their many uses. For example, L-lysine and L-threonine, L-methionine and L-tryptophan are forage additives, and L-glutamate is an additive to control L-isoleucine and L-tyrosine in the pharmaceutical industry, and L-arginine and L Isoleucine is a drug, and L-glutamate, L-aspartate and L-phenylalanine are required as starting materials for the synthesis of fine chemicals.

A method for producing other amino acids is preferably produced biologically using microorganisms, in which a simple and inexpensive raw material is used to obtain each amino acid in a biologically effective and optically active form. For example, in addition to E. coli and related bacteria, Corynebacterium glutamicum and the derivatives Flabac and Lactofermentum (Liebl et al., Int J System Bacteriol 1991, 41: 255 to 260) are used. The bacteria produce amino acids, but only in amounts necessary for growth, making it impossible to produce or recover residual amino acids. This is because the synthesis of amino acids in cells is regulated in many ways. Thus, various methods of increasing product formation by reducing regulatory mechanisms are already known. For example, amino acid derivatives are introduced in this process to block the efficient regulation of biosynthesis. For example, processes resistant to L-tyrosine derivatives and L-phenylalanine derivatives have been used (JP 19037/1976 and 39517/1978). In order to control the regulatory mechanism, a process is used in which bacteria resistant to L-lysine derivatives or L-phenylalanine derivatives are used (EP 0 205 849, GB 2 152 509).

In addition, microorganisms made by recombinant DNA technology and circumventing biosynthetic regulation have been cloned and expressed in genes encoded in the main enzyme that are no longer feedback suppressed. For example, recombinant L-lysine producing bacteria with aspartate kinases, which are feedback resistant that the plasmid encodes, are known (EP 0 381 527). In addition, L-phenylalanine producing bacteria recombined with feedback resistant prephenate dehydrogenase are shown (JP 123475/1986, EP 0 488 424).

In addition, amino acid yield can be increased by overexpressing genes that do not encode enzymes that are likely to respond to feedback during amino acid synthesis. For example, it is possible to form large amounts of lysine by increasing the dihydrodipicolinate synthesis (EP 0 197 335). Many threonine dehydratase can be synthesized to form many isoleucines (EP 0 436 886).

Further research on increasing amino acid production has targeted the improved utility of cellular primary metabolites in central metabolism. Overexpression of transketolase by recombinant technology can increase the formation of L-tryptophan or L-tyrosine or L-phenylalanine (EP 0 600 463). In addition, reducing phosphoenolpyruvate carboxylase activity in Corynebacterium can increase aromatic amino acid formation (EP 0 3331145) while phosphoenolpyruvate carboxylase activity in Corynebacterium Increasing can increase amino acid separation in the aspartate family (EP 0 358 940).

During growth, especially under amino acid production conditions, the tricarboxylic acid cycle must be supplemented with oxalic acetate to replace C4 compounds, for example intermediates recovered for amino acid biosynthesis, continuously and effectively. Until recently pyruvate carboxylase activity has been found in penetrating cells of Corynebacterium glutamicum (Peters-Wendisch et al., Microbiology 1997, 143: 1095 to 1103). This enzyme is inhibited by AMP, ADP, and acetyl coenzyme A, and large amounts are formed when lactate is present as a source of carbon. Some pyruvate carboxylase of Corynebacterium glutamicum has been identified by ¹³ C-NMR spectroscopy and GS-MS (Park et al., Appl. Microbiol. Biotechnol. 1997. 47: 430- 440).

Since this enzyme is determined to satisfy increasing tricarboxylic acid cycles, it is expected that the amino acids belonging to aspartate will not increase with increased gene expression or enzyme activity. In addition, increased gene expression and the enzyme activity of pyruvate carboxylase were not expected to affect the production of other amino acids.

It has been shown that genetic modification of the enzyme increases pyruvate-carboxylase activity and increases pyruvate-carboxylase gene expression, resulting in increased microbial production of aspartate and glutamate family of amino acids. High copy number strains of pyruvate-carboxylase can produce at least 50% lysine, at least 40% threonine, at least 150% homoserine in the culture medium. In addition, glutamate formation was also increased (compare the example in Table 4).

Genetic alteration of pyruvate-carboxylase to increase enzymatic activity can be achieved by altering endogenous genes. Such alterations can be accomplished by classical methods such as UV irradiation or by chemicals that cause the alteration or by genetic techniques such as removal, insertion, or nucleotide exchange.

Increasing gene copy number or enhancing regulatory factors affecting gene expression increases pyruvate-carboxylase gene expression. Thus, preferably, as the transcription signal is increased in the transcription plane, the regulatory element is strengthened. For example, to enhance the effect, gene expression is increased by altering or substituting the promoter sequence of the preceding promoter of the structural gene with a more effective promoter. It affects regulatory genes associated with pyruvate-carboxylase to enhance transcription. For example, mutating a regulatory gene sequence to effect the binding of regulatory proteins to the DNA of the pyruvate-carboxylase gene enhances transcription and enhances gene expression. In addition, the pyruvate-carboxylase gene can be linked with so-called "enhancers" as regulatory sequences and can interchange genes of RNA polymerase and DNA to enhance gene expression of pyruvate-carboxylase. However, for example, the stability of m-RNA is improved and thus the translation is enhanced.

To increase the gene copy number, the pyruvate-carboxylase gene is provided into a gene construct or vector. Genetic constructs include regulatory sequences, particularly those associated with pyruvate-carboxylase genes, preferably regulatory sequences that enhance gene expression. In order to supply the pyruvate-carboxylase gene to the gene construct, the gene was continuously isolated from the Corynebacterium microbial line, and the gene was isolated from the microbial line producing the amino acid, Corynebacterium, Escherichia coli, and Serratia masencins. Transformed. In the method of the present invention, Corynebacterium glutamicum or Corynebacterium glutamicum clamb or Corynebacterium glutamicum lactofermentum is suitable. Gene isolation and in vector and ex vivo recombination (e.g. Simon et al., Bio / Technology 1983, 1: 784 to 791; Eikmanns et al., Gene 1991, 102: 93 to 98), transformation produces amino acids Electrophoresis (Liebl et al., FEMS Microbiology Letters 1991, 65: 299 to 304) or conjugation (Schafer et al., J. Baceriol 1990, 172: 1663 to 1666).

Amino acid producers preferred as host cells use those that are not regulated in the synthesis of the corresponding amino acids or that the activity of the transport carrier for the corresponding amino acids is increased. In addition, strains containing a plurality of central metabolites of the same kind as participated in the synthesis of the corresponding amino acids or small amounts of central metabolites that do not participate in the synthesis of the corresponding amino acids, in particular those containing resistant to competition reactions. Strains are preferred; In other words, a strain that acts with low activity by a synthetic route competitive with the corresponding amino acid biosynthetic pathway is preferred. Therefore, Corynebacterium forming microorganism strains having low citrate synthesis activity are particularly suitable because strains resistant to L-aspartic acid-β-methyl ester (AME) are suitable (EP 0 551 614).

After separation, pyruvate-carboxy having the amino acid sequence described in [SEQ ID NO: 2] or the nucleotide sequence encoding these allelic modifications, or the nucleotide sequences of nucleotides 165 to 3587 described in [SEQ ID NO: 1], or a DNA sequence having substantially the same effect Laase genes can be obtained. The protein promoter further comprises a nucleotide sequence of nucleotides 20 to 109 described in [SEQ ID NO: 1] or a DNA sequence having substantially the same effect. Allelic modifications or DNA sequences of substantially the same effect include functional modifications corresponding to the nucleotide sequences formed by deletion, insertion, or substitution of nucleotides, thereby enabling to maintain or further increase enzymatic activity or function.

This pyruvate-carboxylase gene is preferably used in the method of the present invention. Genes with or without prior promoters and / or pyruvate-carboxylase genes with or without associated regulatory genes are present before or after the DNA sequence and thus are included in the gene structure.

Pyruvate-a promoter (lacI ^O -Gen) - carboxy la azepin chosen which are associated with the modulation sequence in front of the gene.

The pyruvate-carboxylase gene can be cloned to obtain plasmids that contain the gene and are also suitable for transformation with amino acid generators. The transformed cells corresponding to the transformed cells of Corynebacterium comprise genes that can be replicated, i.e., present as additional copies on the chromosome, wherein the copies of the genes are recombined at a selective location in the genome or plasmid or vector. Are combined.

1. Cloning of the Pyruvate-carboxylase Gene of Corynebacterium glutamicum

Saccharomyces cerevisiae (J. Biol Chem 1988, 263: 11493-11497; Mol Gen Genet 1991, 229: 307-315), Mensch (Biochem Biophys Acta 1994, 1227: 46-52), mice (Proc Natl Acad Sci, USA 1993, 90: 1766-1770), Mycobacterium and the conserved regions of all known pyruvate-carboxylase- (pyc-) such as EMBL-GeneBank: Accession Nr. L36530 Starting from tuberculosis (EMBL-GeneBank: Accession Nr. U00024), PCR primers were prepared. The primers correspond to bases 810-831 and 1015-1037 of the pyc gene of Mycobacterium tuberculosis. The chromosomal DNA of Corynebacterium glutamicum ATCC 13032 presented by Eikmanns using PCR for non-productive identical primers according to Innis et al. (PCR protocols.A Guide to Methods and Applications, 1990, Academic press). About 200 bp fragment was isolated by amplification method. 200 bp corresponds to the pyc gene size. The base sequence of the PCR product (Proc Natl Acad Sci USA 1977, 74: 5463-5467) presented by Sanger et al. Was determined. The sequencing was performed by fluorescently labeled ddNTPs using an automated DNA sequencing tool. Starting from the DNA fragment of Corynebacterium glutamicum, the following homologous oligonucleotides can be made.

                 pyc 1 5'- CGTCTTCATCGAAATGAAC- 3 '

                 pyc 2 5'- ACGGTGGTGATCCGGCACT- 3 '

Oligonucleotides are used as PCR primers when separating probes for pyruvate-carboxylase gene (pyc) from Corynebacterium glutamicum. Corynebacterium glutamicum is introduced into a PCR reaction that reacts chromosomal DNA with a dioxygenin labeled nucleotide. The reaction was performed according to the instructions of Boehringer Mannheim's PCR DIG Labeling kits. In this way, about 200 bp of dioxygenin labeled DNA fragment was amplified. The generated pyc probe is used to identify DNA fragments on the chromosomal DNA of Corynebacterium glutamicum in which the pyc gene is located using Southern blot hybrid method. For this purpose, 2-5 μg of wild-type chromosomal DNA of Corynebacterium glutamicum was digested with restriction enzymes HindIII, Sph I, Sal I, Ddra I, EcoR I, and BamH I, and the obtained DNA fragments were reduced to 20 volts in 0.8% agarose gel. It was separated by size by electrophoresis treatment for time. DNA fragments found on agarose gels were denatured to Southern blots (J Mol Biol 1975, 98: 503-517) and Pharmacia LKB was transferred from a genetic matrix onto a nylon membrane (Nytran N13 of Schleicher and Schull, Dassel, Switzerland). Digoxigenin markers, which were fixed by vacuum separation using VacuGene blots and fixed, and also recognized by NBT / X phosphate conversion with alkali, are phosphorylated in this manner. The following chromosomal fragments, 17 kb HindIII fragments, 6.5 kb Sal I fragments and 1.35 kb EcoR I fragments, were hybridized with pyc-DNA-probe.

17 kb HindIII fragments were isolated and subcloned. For this purpose, the chromosomal gene bank of chromosomal DNA of Corynebacterium glutamicum at cosmid pH C79, representing up to 99% of the genome of Corynebacterium glutamicum, was used (Mol Microbiol 1992, 6: 317- 326). Escherichia coli strain DH5α was transformed using this gene bank by the CaCl ₂ method of Sambrook et al. (Molecular cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratories Press) and 300 colonies per LB-agar plate at 50 μg / L. This is very flat. The resulting transformed product was transferred to a nylon N13 filter and incubated for 5 minutes for alkali lysis of cells and alkali lysis and denaturation of DNA occurred in Whatman paper moistened with 0.5 M NaOH and 1.5 M NaCl. Neutralization was then performed with 1M Tris / HCl pH 7.5 and 1.5 M NaCl.

After incubating the filter in 2 × SSC, the released DNA is fixed in the filter by UV irradiation at 366 nm. Remaining cell fragments are removed by shaking in 3 × SSC, 0.1% SDS at 50 ° C. This type of filter was identified by the pyc probe hybrid method as presented by Southern (J Mol Biol 1975, 98: 503-517). Cosmid DNA from this transformant was isolated by plasmid ratio according to the alkaline fusion method of Birnboim (Meth Enzymol 1983, 100: 243-255) and verified by restriction enzyme analysis and Southern blot analysis for the presence of HindIII fragments. Cosmid pH C79-10 contained all 40 kb of HindIII transition and was further analyzed. After restriction enzyme treatment with the endonuclease SalI and EcoRI, hybrid fragments identical to chromosomal DNA, that is, 6.5 kb SalI fragment and 1.35 kb EcoRI fragment, appeared. The 17 kb HindIII fragment was isolated from cosmid by restriction enzyme treatment and ligated into E. coli vector pUC 18 and also split into HindIII. The resulting fragment of the vector pUC pyc was analyzed for restriction enzymes. The genetic map of the fragment is shown in FIG. It is presented in 1.

2. Determination of the base sequence of pyruvate-carboxylase gene

In subsequent subcloning steps, 0.85 kb SalI-EcoRI fragments were treated from the plasmid pUC pyc by treatment with corresponding restriction enzymes such as 1.35 kb EcoRI fragments, 1.6 kb EcoRI-EcoRI-StuI fragments and 1.6 kb ClaI fragments that overlap with this fragment. Separated. The fragments were cloned into restriction vector pUC18 by ligation and then sequenced according to the method suggested by Sanger et al. (Proc Natl Acad Sci USA 1977, 74: 5463-5467). The obtained nucleotide sequence was analyzed according to the program package HUSAR (Release 3.0) (Heidelberg) in Germany for cancer research. Analysis of the nucleotide sequence of the fragment revealed a 3576 bp encoding structure encoding the protein sequence of 1140 amino acids. Comparing the protein sequence with the EMBL gene information bank (Heidelberg), it can be seen that there is similarity with the known pyruvate carboxylase. It was most similar to the estimated pyruvate carboxylase of mycobacterium tuberculosis (EMBL-GeneBank: Accession No. U00024) (62%). The similarity reaches 76% when the exchange of conserved amino acids takes place. Compared to pyruvate carboxylase of other organisms, the amino acids were 46-47% identical and 64-65% similar. (Gene 1997,191: 47-50; J Bacteriol 1996, 178: 5960-5970; Proc Natl Acad Sci USA 1993, 990: 1766-1770; Biochem J 1996, 316: 631-637; EMBL-GenBank: Accession No. L 36530; J Biol Chem 1988, 263: 11493-11497; Mol Gen Genet 1991, 229: 307-315). As a result, the cloned fragment base was confirmed to be the pyruvate carboxylase gene of Corynebacterium glutamicum. The nucleotide sequence of this gene is as described in [SEQ ID NO: 1], and the corresponding amino acid sequence is as described in [SEQ ID NO: 2].

3. Overexpression of Pyruvate Carboxylase

 For overexpression of the pyruvate carboxylase gene of Corynebacterium glutamicum, the gene was cloned from plasmid pUCpyc into 6.2 kb SspI-ScaI-fragment of E. coli. Ends treated with cleno-polymerase were ligated to the smooth ends by suturing EcoRI or binding pstI, and linear vectors were ligated into 6.2 kb SspI-ScaI-fragments. The resulting construct, pEK0pyc, was transformed in E. coli strain DH5α, plasmid DNA was isolated from the transformants, and the accuracy of the insert was controlled by restriction enzyme treatment. DNA was introduced into strain SP 733 by electrophoresis (FEMS Microbiol Lett 1989, 65: 299-304).

This strain is a variant of the limited negative Corynebacterium glutamicum strain R127 (Dechema Biotechnology Conference 1990, 4: 323-327, Verlag Chemie) and strain R127 was obtained by chemical mutagenesis, with pyruvate and lactate being the only carbon Minimal medium of origin had properties that could not be grown (Microbiology 1997, 143: 1095-1103). This phenotype is recognized as a deficiency of pyruvate carboxylase and is derived from a strain that contrasts with the starting strain, which is carried by C. glutamicum, ie plasmid pEK0pyc, and can be grown again in the presence of a minimal medium with lactate, the only carbonaceous park. The derived pyruvate carboxylase gene can be introduced and supplemented. This fact verifies that the gene encodes pyruvate carboxylase.

In addition, the plasmid pEK0pyc was transformed from Corynebacterium glutamicum wild type ATCC 13032 by electrophoresis. Wild type strain pEK0pyc was compared with wild type ATCC 13032 for pyruvate carboxylase activity. Strains were cultured in a complex medium containing 0.5% lactate (Luria-Bertani, Molecular Cloning, A laboratory mannual, 1989, Cold Spring Harbor Laboratory Press) and in a minimal medium containing 2% lactate or 4% glucose, Peters-Wendisch et al. Pyruvate carboxylase activity was tested according to this suggested method. As a result, the pyruvate carboxylase activity of the strain with pEK0-pyc was four times higher than that of the starting strain.

4. Accumulation of lysine by overexpression of pyruvate carboxylase gene in strain Corynebacterium glutamicum DG 52-5

To investigate the effect of overexpression of the pyruvate carboxylase gene in lysine producing strain DG52-5 (J Gen Microbiol 1988,134: 3221-3229), the expression vector pVWEX1 was used to promote IPTG-induced expression. . In this vector, the pyc gene was cloned without the promoter. For this purpose, PCR-primers (primer 1 = position 112-133; primer 2 = position 373-355) were synthesized from the nucleotide sequence according to [SEQ ID NO: 1] and the promoter-free starting region 261bp of the pyruvate carboxylase gene Was amplified by PCR.

Primer 1 was selected to enable the PstI cleavage site and Primer 2 to enable the BamH I cleavage site. After PCR, the 274bp PCR product was isolated and ligated into the concamer, and divided into restriction enzymes PstI and BamHI. The restriction enzyme product is concentrated by ethanol precipitation and ligated into the PstI-BamHI split vector pVWEX1. The construct pVWEX®-PCR thus formed was tested by restriction enzyme treatment. The terminal of the pyc gene was isolated from the vector pEK0pyc by RcaI-Klenow-SalI treatment and ligated to BamHI-Klenow-SalI in the vector pVWEX1-PCR process. The structure pVWEX1pyc thus formed was analyzed by restriction guidance. A map of the plasmid is shown in FIG.

The plasmid was introduced into Corynebacterium glutamicum DG 52-5 by electrophoresis. As a control, strain DG 52-5 was transformed with the vector pVWEX1 without inserts and the precipitation of L-lysine of the three transformants was compared. For this purpose, each fermentation medium was independently inoculated from DG 52-5 (pVWEX1pyc) 3,4 and 2xTY (Molecular cloning, A laboratory manual, 1989, Cold Spring Harbor Laboratory Press with 50 μg / L kanamycin) and previous stage medium. The medium further contains kanamycin to stabilize the plasmid. In each case, 200 μg IPTG / ml was added to one flask while the second flask performed two equivalent experiments without IPTG. The amount of lysine accumulated in the medium was determined after incubation at 120 RPM in a rotary shaker for 48 hours at 30 ° C. Amino acid concentration was determined by high pressure liquid chromatography (J Chromat 1983, 266; 471-482).

The fermentation results are shown in Table 2 and the values given are the average of three experiments with different clones. Overexpression of the pyruvate carboxylase gene resulted in 50% more lysine accumulation in the medium. The gene of the preservative enzyme pyruvate carboxylase further enhances lysine formation.

5. Accumulation of threonine and homoserine by high expression of pyruvate carboxylase gene in strain Corynebacterium glutamicum DM 368-3

Similar to the experiments on L-lysine formation, the accumulation of threonine in the culture supernatants due to the high expression of the pyruvate carboxylase gene was also investigated for this purpose, as suggested in Section 4. The threonine producing strain Corynebacterium glutamicum DM 368-3 (Degussa AG) was transformed with plasmid pVWEX1pyc and transformed with plasmid pVWEX1 as a control and three different transformants were analyzed for threonine isolation. For this purpose, DM368-3 (pVWEX1) 2 and 3, DM 368-3 (pVWEX1pyc) 1,2,3 were incubated in complex medium (2xTY with 50 μg / L kanamycin) and fermented medium CGXII (J Bacteriol in each case). 1993, 175: 5595-5603) were each inoculated in preculture. The medium further comprises kanamycin to stabilize the plasmid. One flask was added with 200 μg IPTG / ml, whereas the second flask performed two equivalent experiments in the form without IPTG. The amount of threonine accumulated in the medium was determined after incubation at 120 RPM in a rotary shaker for 48 hours at 30 ° C. Amino acid concentration was determined by high pressure liquid chromatography (J Chromat 1983, 266; 471-482). The fermentation results are shown in Table 3 and the values given are the average of three experiments with different clones. Overexpression of the pyruvate carboxylase gene resulted in 40% more threonine accumulation in the medium. L-threonine formation is enhanced by using the gene of the proposed complement enzyme pyruvate carboxylase during L-threonine formation.

In addition, it can be seen that strains overexpressed with the pyruvate carboxylase gene by amino acid concentration determination produced 150% more homoserine than strains with non-overexpressed genes. The results are shown in Table 3. In the process according to the invention it can be seen that the formation of threonine, such as homoserine is greatly improved.

6. Accumulation of glutamate by overexpression of pyruvate carboxylase gene in Corynebacterium glutamicum wild type

Similar to the experiments for L-lysine, L-threonine and L-homoserine formation (see Sections 4 and 5 above), the accumulation of glutamate in the culture supernatant by overexpression of the pyruvate carboxylase gene was investigated. For this purpose, Corynebacterium glutamicum wildtype with plasmid pVWEX1 was transformed and glutamate isolation was determined from two different transformants in addition to the control with plasmid pVWEX1 as indicated. Corynebacterium glutamicum ATCC 13032 (pVWEX1pyc) 1 and 2 together with Corynebacterium glutamicum ATCC 13032 pVWEX1pyc D1 and D2 were incubated in complex medium (2xTY with 50 μg / l kanamycin) Fermentation medium CGXII (J Bacteriol 1993, 175: 5595-5603) was inoculated during each preculture period. The medium further contains kanamycin to stabilize the plasmid. Six hours after inoculation, 60 mg at 25 mg per ml was added to the medium to induce glutamate isolation. One flask was added with 200 μg IPTG / ml while the second flask performed two equivalent experiments without IPTG. The amount of threonine accumulated in the medium was determined after incubation at 120 RPM in a rotary shaker for 48 hours at 30 ° C. Amino acid concentration was determined by high pressure liquid chromatography (J Chromat 1983, 266; 471-482). The fermentation results are shown in Table 4 and the values given are the average of three experiments with different clones. Overexpression of the pyruvate carboxylase gene increased the concentration of glutamate in the medium by 200%. The use of the gene of the proposed complement enzyme pyruvate carboxylase improves glutamate formation.

In addition, it can be seen that strains overexpressing the pyruvate carboxylase gene produced 150% more homoserine than strains with non-overexpressed genes. The results are shown in Table 3. In the process according to the invention it can be seen that the formation of threonine, such as homoserine is greatly improved.

 Table 1

Strain IPTG Pyruvate carboxylase [Μg / ml] [nmol min ^-1 mg, anhydrous weight ^-1 ] 13032 (pEK0pyc) 0 75 ± 13 ATCC 13032 0 19 ± 4 DG52-5 (pVWEX1pyc) 200 88 ± 13 0 11 ± 2 DG52-5 (pVWEX1) 200 5 ± 2 0 6 ± 1 DM368-3 (pVWEX1pyc) 200 76 ± 10 0 12 ± 3 DM368-3 (pVWEX1) 200 10 ± 1 0 11 ± 2

Table 2

Strain IPTG Lysine [Μg / ml] [mM] DG52-5 (pVWEX1pyc) 200 35.4 ± 2.6 0 23.6 ± 2.9 DG52-5 (pVWEX1) 200 23.3 ± 2.9 0 22.1 ± 4.0

Table 3

Strain IPTG Threonine Homoserine [Μg / ml] [mM] [mM] DM368-3 (pVWEX1pyc) 200 10.2 ± 0.5 14.4 ± 1.2 0 7.9 ± 1.0 5.6 ± 0.2 DM368-3 (pVWEX1) 200 8.0 ± 0.5 5.8 ± 0.7 0 7.5 ± 0.8 6.1 ± 1.0

Table 4

Strain IPTG Glutamate [Μg / ml] [mM] ATCC 13032 200 11 ± 2 ATCC 13032 0 13 ± 2 ATCC 13032 (pVWEX1-pyc) 200 67 ± 4 ATCC 13032 (pVWEX1-pyc) 0 32 ± 4

<110> FORSCHUNGSZENTRUM JULICH GMBH <120> METHOD FOR MICROBIAL PRODUCTION OF AMINO ACIDS OF THE ASPARATATE AND / OR GLUTAMATE FAMILY AND .. <150> DE 197 43 894.6 <151> 1997-10-04 <160> 2 <170> KOPATIN 1.5 <210> 1 <211> 3728 <212> DNA <213> Corynebacterium glutamicum <400> 1 cgcaaccgtg cttgaagtcg tgcaggtcag gggagtgttg cccgaaaaca ttgagaggaa 60 aacaaaaacc gatgtttgat tgggggaatc gggggttacg atactaggac gcagtgactg 120 ctatcaccct tggcggtctc ttgttgaaag gaataattac tctagtgtcg actcacacat 180 cttcaacgct tccagcattc aaaaagatct tggtagcaaa ccgcggcgaa atcgcggtcc 240 gtgctttccg tgcagcactc gaaaccggtg cagccacggt agctatttac ccccgtgaag 300 atcggggatc attccaccgc tcttttgctt ctgaagctgt ccgcattggt accgaaggct 360 caccagtcaa ggcgtacctg gacatcgatg aaattatcgg tgcagctaaa aaagttaaag 420 cagatgccat ttacccggga tacggcttcc tgtctgaaaa tgcccagctt gcccgcgagt 480 gtgcggaaaa cggcattact tttattggcc caaccccaga ggttcttgat ctcaccggtg 540 ataagtctcg cgcggtaacc gccgcgaaga aggctggtct gccagttttg gcggaatcca 600 ccccgagcaa aaacatcgat gagatcgtta aaagcgctga aggccagact taccccatct 660 ttgtgaaggc agttgccggt ggtggcggac gcggtatgcg ttttgttgct tcacctgatg 720 agcttcgcaa attagcaaca gaagcatctc gtgaagctga agcggctttc ggcgatggcg 780 cggtatatgt cgaacgtgct gtgattaacc ctcagcatat tgaagtgcag atccttggcg 840 atcacactgg agaagttgta cacctttatg aacgtgactg ctcactgcag cgtcgtcacc 900 aaaaagttgt cgaaattgcg ccagcacagc atttggatcc agaactgcgt gatcgcattt 960 gtgcggatgc agtaaagttc tgccgctcca ttggttacca gggcgcggga accgtggaat 1020 tcttggtcga tgaaaagggc aaccacgtct tcatcgaaat gaacccacgt atccaggttg 1080 agcacaccgt gactgaagaa gtcaccgagg tggacctggt gaaggcgcag atgcgcttgg 1140 ctgctggtgc aaccttgaag gaattgggtc tgacccaaga taagatcaag acccacggtg 1200 cagcactgca gtgccgcatc accacggaag atccaaacaa cggcttccgc ccagataccg 1260 gaactatcac cgcgtaccgc tcaccaggcg gagctggcgt tcgtcttgac ggtgcagctc 1320 agctcggtgg cgaaatcacc gcacactttg actccatgct ggtgaaaatg acctgccgtg 1380 gttccgactt tgaaactgct gttgctcgtg cacagcgcgc gttggctgag ttcaccgtgt 1440 ctggtgttgc aaccaacatt ggtttcttgc gtgcgttgct gcgggaagag gacttcactt 1500 ccaagcgcat cgccaccgga ttcattgccg atcacccgca cctccttcag gctccacctg 1560 ctgatgatga gcagggacgc atcctggatt acttggcaga tgtcaccgtg aacaagcctc 1620 atggtgtgcg tccaaaggat gttgcagctc ctatcgataa gctgcctaac atcaaggatc 1680 tgccactgcc acgcggttcc cgtgaccgcc tgaagcagct tggcccagcc gcgtttgctc 1740 gtgatctccg tgagcaggac gcactggcag ttactgatac caccttccgc gatgcacacc 1800 agtctttgct tgcgacccga gtccgctcat tcgcactgaa gcctgcggca gaggccgtcg 1860 caaagctgac tcctgagctt ttgtccgtgg aggcctgggg cggcgcgacc tacgatgtgg 1920 cgatgcgttt cctctttgag gatccgtggg acaggctcga cgagctgcgc gaggcgatgc 1980 cgaatgtaaa cattcagatg ctgcttcgcg gccgcaacac cgtgggatac accccgtacc 2040 cagactccgt ctgccgcgcg tttgttaagg aagctgccag ctccggcgtg gacatcttcc 2100 gcatcttcga cgcgcttaac gacgtctccc agatgcgtcc agcaatcgac gcagtcctgg 2160 agaccaacac cgcggtagcc gaggtggcta tggcttattc tggtgatctc tctgatccaa 2220 atgaaaagct ctacaccctg gattactacc taaagatggc agaggagatc gtcaagtctg 2280 gcgctcacat cttggccatt aaggatatgg ctggtctgct tcgcccagct gcggtaacca 2340 agctggtcac cgcactgcgc cgtgaattcg atctgccagt gcacgtgcac acccacgaca 2400 ctgcgggtgg ccagctggca acctactttg ctgcagctca agctggtgca gatgctgttg 2460 acggtgcttc cgcaccactg tctggcacca cctcccagcc atccctgtct gccattgttg 2520 ctgcattcgc gcacacccgt cgcgataccg gtttgagcct cgaggctgtt tctgacctcg 2580 agccgtactg ggaagcagtg cgcggactgt acctgccatt tgagtctgga accccaggcc 2640 caaccggtcg cgtctaccgc cacgaaatcc caggcggaca gttgtccaac ctgcgtgcac 2700 aggccaccgc actgggcctt gcggatcgtt tcgaactcat cgaagacaac tacgcagccg 2760 ttaatgagat gctgggacgc ccaaccaagg tcaccccatc ctccaaggtt gttggcgacc 2820 tcgcactcca cctcgttggt gcgggtgtgg atccagcaga ctttgctgcc gatccacaaa 2880 agtacgacat cccagactct gtcatcgcgt tcctgcgcgg cgagcttggt aaccctccag 2940 gtggctggcc agagccactg cgcacccgcg cactggaagg ccgctccgaa ggcaaggcac 3000 ctctgacgga agttcctgag gaagagcagg cgcacctcga cgctgatgat tccaaggaac 3060 gtcgcaatag cctcaaccgc ctgctgttcc cgaagccaac cgaagagttc ctcgagcacc 3120 gtcgccgctt cggcaacacc tctgcgctgg atgatcgtga attcttctac ggcctggtcg 3180 aaggccgcga gactttgatc cgcctgccag atgtgcgcac cccactgctt gttcgcctgg 3240 atgcgatctc tgagccagac gataagggta tgcgcaatgt tgtggccaac gtcaacggcc 3300 agatccgccc aatgcgtgtg cgtgaccgct ccgttgagtc tgtcaccgca accgcagaaa 3360 aggcagattc ctccaacaag ggccatgttg ctgcaccatt cgctggtgtt gtcaccgtga 3420 ctgttgctga aggtgatgag gtcaaggctg gagatgcagt cgcaatcatc gaggctatga 3480 agatggaagc aacaatcact gcttctgttg acggcaaaat cgatcgcgtt gtggttcctg 3540 ctgcaacgaa ggtggaaggt ggcgacttga tcgtcgtcgt ttcctaaacc tttctgtaaa 3600 aagccccgcg tcttcctcat ggaggaggcg gggctttttg ggccaagatg ggagatgggt 3660 gagttggatt tggtctgatt cgacactttt aagggcagag atttgaagat ggagaccaag 3720 gctcaaag 3728 <210> 2 <211> 1140 <212> PRT <213> Corynebacterium glutamicum <400> 2 Met 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 Lys 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 Xaa 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 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

Claims (37)

A method for microbiological preparation of aspartate and / or glutamate family of amino acids, characterized in that pyruvate carboxylase activity is increased by genetic modification of enzymes and / or pyruvate carboxylase gene expression of amino acid-forming microorganisms. The method of claim 1, A method for the microbial preparation of amino acids, characterized in that the high pyruvate carboxylase active gene is produced by mutation of the endogenous pyruvate carboxylase gene. The method according to claim 1 or 2, Microbial preparation of amino acids characterized by increasing the number of gene copies to increase the gene expression of pyruvate carboxylase The method of claim 3, wherein Microbial preparation method of amino acids characterized in that the gene of pyruvate carboxylase is bound to the gene construct in order to increase the number of gene copies The method of claim 3, wherein A microbiological method for producing an amino acid, characterized in that the pyruvate carboxylase gene is coupled to a gene construct comprising a regulatory gene sequence associated with the gene of pyruvate carboxylase. The method according to claim 4 or 5, A microbial method for producing an amino acid, characterized in that the amino acid-producing microorganism is transformed by the gene-containing gene construct. The method of claim 6, Corynebacterium sp. Microorganism is a microbial method for producing an amino acid, characterized in that the transformed by the gene-containing gene construct. The method of claim 6, A method for producing a microbial amino acid, characterized by using a microorganism which inhibits the regulation of an enzyme participating in the corresponding amino acid synthesis for transformation and / or improves the activity of an external carrier of the corresponding amino acid. The method of claim 6, A method for producing a microbial amino acid, characterized by using a microorganism containing a large amount of the central metabolite of the corresponding amino acid participating in the synthesis for transformation. The method of claim 6, A method for producing a microbial amino acid, characterized by using a microorganism having a biosynthetic pathway that proceeds with low activity and competes with a biosynthetic pathway of a corresponding amino acid for transformation. The method of claim 1, A method for producing microorganisms of amino acids, wherein the pyruvate carboxylase gene is isolated from various kinds of Corynebacterium microbial strains. The method of claim 1, A method for producing a microbial amino acid, characterized in that to enhance the transcription signal to increase gene expression. The method of claim 1, A microbiological method for producing an amino acid, characterized in that the pyruvate carboxylase gene has a tac-promoter in front of the pyruvate carboxylase gene. The method of claim 13, The tack promoter is a microbiological method for producing an amino acid, characterized in that it is linked to a regulatory sequence. The method of claim 1, A pyruvate carboxylase gene is a microbial method for producing an amino acid, characterized in that the gene having the amino acid sequence described in [SEQ ID NO: 2] and a nucleotide sequence encoding an allelic modification thereof.        The method of claim 15,        A microbiological method for producing an amino acid, comprising using a gene having a nucleotide sequence of nucleotides 165 to 3587 described in [SEQ ID NO: 1] or a DNA sequence having substantially the same effect together with a pyruvate carboxylase gene.       The method of claim 1,      A method for producing microorganisms of amino acids, characterized by producing lysine, threonine, homoserine, glutamate and / or arginine. A pyruvate carboxylase gene characterized by having a nucleotide sequence encoding the amino acid sequence described in [SEQ ID NO: 2] and / or encoding an allelic modification thereof. The method of claim 18, A pyruvate carboxylase gene characterized by having a nucleotide sequence of nucleotides 165 to 3587 or a DNA sequence having substantially the same effect as described in [SEQ ID NO: 1]. The method of claim 18, A pyruvate carboxylase gene, characterized by having a preceding promoter of the nucleotide sequence of nucleotides 20 to 109 described in [SEQ ID NO: 1] or a DNA sequence having substantially the same effect. The method of claim 18 or 19, A pyruvate carboxylase gene, characterized by having a preceding tack promoter. The method of claim 21, A pyruvate carboxylase gene, characterized by having a regulatory gene sequence associated with a promoter.        The method according to any one of claims 18 to 20, A pyruvate carboxylase gene characterized by having a regulatory gene sequence associated with these sequences. A gene structure comprising the pyruvate carboxylase gene of claim 18. A vector comprising a pyruvate carboxylase gene according to claim 18 or a gene structure according to claim 24. A transformed cell comprising a pyruvate carboxylase gene according to claim 18 or a gene structure according to claim 24 in cloned form. The method of claim 26, A transformed cell comprising the vector according to claim 25. The method of claim 26 or 27, Transformed cells characterized by belonging to various types of Corynebacterium. The method of claim 26, Wherein the regulation of enzymes participating in the synthesis of the corresponding amino acids and / or enzymes participating in the external transport of the corresponding amino acids is inhibited. The method of claim 26, A transformed cell, characterized in that it contains a large amount of a central metabolite involved in the synthesis of amino acids. The method of claim 26, A transformed cell, characterized in that it contains a small amount of a central metabolite that does not participate in the synthesis of amino acids. A method of using a pyruvate carboxylase gene, characterized in that it is used to increase the production of aspartate or glutamate amino acids. The method of claim 32, A method of using a pyruvate carboxylase gene, characterized in that a mutant pyruvate carboxylase gene encoding an enzyme of high pyruvate carboxylase activity is used. The method of claim 32 or 33, A microorganism producing a corresponding amino acid is transformed by a gene structure comprising a pyruvate carboxylase gene. The method of claim 34, A method of using a pyruvate carboxylase gene, characterized in that the gene structure further comprises a regulatory gene sequence. The method of claim 32, A method of using pyruvate carboxylase, characterized in that pyruvate carboxylase gene of Corynebacterium is used. The method of claim 32, A method of using pyrubenite carboxylase characterized in that Corynebacterium is used as an amino acid producing microorganism.