RU2418064C2 - METHOD OF OBTAINING L-AMINO ACID OF GLUTAMATE FAMILY, OR L-BALINE WITH APPLICATION OF BACTERIUM BELONGING TO GENUS Escherichia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of obtaining L-amino acid, belonging to glutamate family; or L-valine with application of bacterium, belonging to genus Escherichia, which is modified in such a way that glutamate dehydrogenase activity in said bacteria is increased.

EFFECT: invention makes it possible to obtain L-amino acid, belonging to glutamate family, or L-valine with high degree of efficiency.

6 cl, 1 dwg, 2 tbl, 8 ex

Description

Technical field

The present invention relates to the microbiological industry, in particular to a method for producing an L-amino acid belonging to the glutamate family using a bacterium of the Enterobacteriaceae family, modified in such a way that the activity of glutamate dehydrogenase in said bacterium is increased.

Description of the Related Art

Traditionally, L-amino acids on an industrial scale are obtained by fermentation using strains of microorganisms obtained from natural sources, or their mutants. Typically, microorganisms are modified to increase the production of L-amino acids.

Many methods have been described to increase the production of L-amino acids, including transformation of recombinant DNA microorganisms (see, for example, US Pat. No. 4,278,765). Other methods of increasing production include increasing the activity of enzymes involved in the biosynthesis of amino acids, and / or reducing the sensitivity of the target enzyme to feedback inhibition produced by the L-amino acid (see, for example, international application WO 95/16042 or US patent 4346170; 5661012 and 6040160).

Known DNA sequence of the gdhA gene of Escherichia coli K12 encoding a NADP-specific glutamate dehydrogenase subunit polypeptide consisting of 447 amino acids. The protein sequence corresponding to the gene is substantially homologous to the corresponding enzyme of the fungus Neurospora crassa. The DNA sequence before the gene includes several overlapping typical promoter sequences. The DNA sequence after the gene contains inverted repeats, presumably forming extended stable stem loop loop RNA structures homologous to those found in several intergenic regions of enterobacteria (McPherson M.Y. and Wootton J.C., Nucleic Acids Res .; 11 (15): 5257-66 (1983)).

Glutamate dehydrogenase (GDH - glutamate dehydrogenase) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia. Data on GDH-specific activity using excess and insufficient concentrations of ammonia as a source of nitrogen are presented. It is proved that the level of mRNA for a given enzyme depends on the concentration of ammonia in the nutrient medium. A single and apparently constant transcript was determined for several growing conditions. The data on the identification of the functional promoter and the corresponding transcription start point under certain growing conditions are presented. The putative regulatory sequences are discussed localized in the region adjacent to the 5'-end of the gdhA gene (Riba L. et al., Gene; 71 (2): 233-46 (1988)).

Data have been published that the E. coli gdhA gene encoding NADP-specific glutamate dehydrogenase is located on the map in 38.6 min. This localization is confirmed by the shown linkage with three Tn10 inserts that are linked to the aroD, pheS, and ansA loci, complementation of the mapped restriction clone A. and the correspondence between the restriction maps of the chromosome and the cloned and sequenced gdhA gene (Kim SY et al., J Bacteriol .; 172 (10): 6127-8 (1990)).

GDH is absent in Rhizobium phaseoli, and ammonia is assimilated via the glutamine synthetase-glutamate synthase pathway. A strain of R. phaseoli was constructed containing the structural gene GDH (gdhA) from E. coli. GDH activity was expressed in R. phaseoli in the free state and in symbiosis. In nodules with bacteroids that expressed GDH activity, nitrogen fixation was significantly reduced. In addition, R. phaseoli cells lacking GDH activity and assimilating ammonia via the glutamine synthetase-glutamate synthase pathway preferably formed nodules in Phaseolus vulgaris (Bravo A. et al., J Bacteriol .; 170 (2): 985-8 (1988)) .

The structural gene for glutamate dehydrogenase, gdhA, is mapped on the genetic map at 38.6 min and 1860 kb on the physical map. A detailed map of this area is presented (Helling R.B., Mol Gen Genet .; 223 (3): 508-12 (1990)).

It was shown that the E. coli nac gene is transcriptionally active and that its expression is dependent on NRI (NtrC) and σ-54. Northern blot experiments show the presence of monocistronic nac-specific mRNA when growing wild-type cells under a nitrogen limit. Evidence has been obtained that, under the nitrogen limit, Nac is involved in the repression of gdhA gene transcription, unless L-glutamine is used as the sole nitrogen source. In addition, the high level of GDH activity observed in the nac mutant strain was decreased when the wild-type nac gene was expressed under the control of the lac promoter under the nitrogen limit, unless L-glutamine was used as the sole source of nitrogen or in conditions of excess nitrogen . These results suggest the existence of an additional mechanism for overcoming Nac repression (Camarena L. et al., FEMS Microbiol Lett; 167 (1): 51-6 (1998)).

A method is disclosed for producing L-glutamine by culturing a coryneform bacterium capable of producing L-glutamine, modified in such a way that its intracellular glutamine synthetase activity is increased, preferably further modified to enhance intracellular glutamate synthetase activity, in the medium for production and accumulation of L-glutamine and excretion L-Glutamine (EP 1229121 A2).

There are currently no reports describing the use of increased glutamate dehydrogenase activity to increase the production of L-amino acids belonging to the family of glutamate, L-threonine and L-valine.

Description of the invention

Objectives of the present invention include increasing the productivity of strains producing L-amino acids belonging to the glutamate family and providing a method for producing L-amino acids belonging to the family of glutamate, L-threonine and L-valine using these strains.

The above objectives were achieved by discovering that an increase in glutamate dehydrogenase activity can lead to an increase in the production of L-amino acids belonging to the glutamate family, such as L-glutamic acid, L-glutamine, L-proline, L-arginine, ornithine and citrulline; L-threonine and L-valine.

The present invention provides a bacterium of the Enterobacteriaceae family that is capable of increased production of amino acids belonging to the glutamate family, such as L-glutamic acid, L-glutamine, L-proline, L-arginine, ornithine and citrulline; L-threonine and L-valine.

The aim of the present invention is the provision of belonging to the Enterobacteriaceae family of bacteria producing L-amino acids belonging to the family of glutamate, L-threonine or L-valine, modified so that the activity of glutamate dehydrogenase in the bacteria is increased.

It is also an object of the present invention to provide a bacterium as described above in which said activity is enhanced by enhancing expression of the gdhA gene.

It is also an object of the present invention to provide a bacterium as described above in which expression of said gdhA gene is enhanced by increasing the number of copies of a gene or modifying a sequence that controls gene expression, as a result of which gene expression is enhanced.

It is also an object of the present invention to provide the bacteria described above, wherein said bacterium belongs to the genus Escherichia.

It is also an object of the present invention to provide the bacteria described above, wherein said bacterium belongs to the genus Pantoea.

It is also an object of the present invention to provide the bacterium described above, wherein said L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-arginine, ornithine and citrulline.

Another objective of the present invention is the provision of a method for producing L-amino acids belonging to the family of glutamate, L-threonine or L-valine, including:

- growing the above bacteria in a nutrient medium, and

- the allocation of the specified L-amino acids from the culture fluid.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-arginine, ornithine and citrulline.

The present invention is described in detail below.

BEST MODE FOR CARRYING OUT THE INVENTION

1. The bacterium according to the present invention.

A bacterium according to the present invention is a bacterium producing the L-amino acid belonging to the glutamate family belonging to the Enterobacteriaceae family, modified in such a way that the activity of glutamate dehydrogenase in said bacterium is increased.

According to the present invention, “L-amino acid producing bacterium” means a bacterium capable of producing and secreting an L-amino acid into a culture medium when the bacterium of the present invention is grown in said culture medium.

As used herein, the term “L-amino acid producing bacterium” also means a bacterium that is capable of producing and causes the accumulation of an L-amino acid belonging to the glutamate family in a fermentation medium in quantities greater than the natural or parental E. coli strain, such as a strain of E. coli K-12, and preferably means that the microorganism is capable of accumulating in the medium the target L-amino acid in an amount of not less than 0.5 g / l, more preferably not less than 1.0 g / l.

The term “L-amino acid belonging to the glutamate family” includes L-glutamic acid, L-glutamine, L-proline, L-arginine, ornithine and citrulline.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. More specifically, bacteria classified as belonging to the Enterobacteriaceae family according to the taxonomy used in the NCB1 database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/ can be used. wgetorg? mode = Tree & id = 1236 & lvl = 3 & keep = l & srchmode = l & unlock). A bacterium belonging to the genus Escherichia or Pantoea is preferred.

The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, the bacterium Escherichia coli (E. coli) may be mentioned.

The range of bacteria belonging to the genus Escherichia that can be used in the present invention is not limited in any way, however, for example, the bacteria described in Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) can be included among the bacteria of the present invention.

The term "bacterium belonging to the genus Pantoea" means that the bacterium belongs to the genus Pantoea in accordance with the classification known to the specialist in the field of microbiology. Recently, several Enterobacter agglomerans species have been classified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like based on analysis of the 16S rRNA nucleotide sequence, etc. (Int. J. Syst. Bacteriol, 43, 162-173 (1993)).

The term “bacterium is modified in such a way that the activity of glutamate dehydrogenase in the specified bacterium is increased” means that the bacterium is modified in such a way that the activity of glutamate dehydrogenase in the modified bacterium is higher than in the unmodified one. The term "enhancing gdhA gene expression" means that gene expression is higher than in an unmodified strain, for example, a wild-type strain. Examples of such modifications include an increase in the number of copies of an expressed gene in a cell, an increase in the level of gene expression, etc.

The number of copies of the expressed gene is determined, for example, by restriction of the chromosomal DNA followed by Southern blotting using a probe constructed based on the gene sequence, in situ fluorescence hybridization (FISH), and the like. The level of gene expression can be determined by various known methods, including Northern blotting, quantitative RT-PCR, etc. The amount of protein encoded by the gene can be determined using known methods, including SDS-PAGE electrophoresis followed by immunoblotting (Western blotting) and the like.

The gdhA gene (synonyms: ECK1759, b1761) encodes glutamate dehydrogenase (synonym B 1761). The gdhA gene (nucleotides from 1,840,395 to 1,841,738 in sequence with accession number NC_000913.2 in the GenBank database; gi: 16129715) is located between the OPC ynjH and OPC ynjI, oriented in the opposite direction to gdhA, on the chromosome of E. coli K-12 strain. The nucleotide sequence of the gdhA gene and the amino acid sequence of GdhA encoded by the gdhA gene are shown in the Sequence Listing Numbers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively.

Since representatives of various genera or strains of the Enterobacteriaceae family may experience some variations in the nucleotide sequences, the concept of the gdhA gene is not limited to the gene shown in the Sequence Listing under SEQ ID NO: 1, but may also include genes homologous to SEQ ID NO: 1 encoding a variant of the GdhA protein.

The term "protein variant", as used in the present invention, means a protein with changes in sequence, be it deletion, insertion, addition or replacement of amino acids. The number of changes in a protein variant depends on the position or type of amino acid residue in the tertiary structure of the protein. It can be from 1 to 30, preferably from 1 to 15, more preferably from 1 to 5 in SEQ ID NO: 2. These variations in variants may occur in areas not critical to protein function. These changes are possible because some amino acids have a high homology to each other, therefore, such changes do not affect the tertiary structure or activity. Therefore, a variant of the protein encoded by the gdhA gene can have a homology of at least 80%, preferably at least 90% and most preferably at least 95%, with respect to the complete amino acid sequence shown in SEQ ID NO. 2, while maintaining the activity of glutamate dehydrogenase.

Homology between two amino acid sequences can be determined using known methods, for example, the BLAST 2.0 computer program, which counts three parameters: amino acid number, identity, and similarity.

In addition, the ydbK gene may be a variant that hybridizes under stringent conditions with the nucleotide sequence shown in the Sequence Listing under the number SEQ ID NO: 1, or with a probe that can be synthesized based on the specified nucleotide sequence, provided that before inactivation it encodes a functional protein YdbK. "Stringent conditions" include those conditions under which specific hybrids, for example hybrids with a homology of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% and most preferably at least 95%, are formed, and non-specific hybrids, for example hybrids with less homology than indicated above, are not formed. A practical example of harsh conditions is a single or multiple washing, preferably two or three times, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N + nylon membrane (Amersham) under stringent conditions is 15 minutes. Two to three times washing is preferred. The length of the probe can be selected depending on the hybridization conditions, in this particular case, it can be about 100-1000 bp.

The following discloses a method for increasing glutamate dehydrogenase activity. Using the Escherichia coli gene, the gene encoding glutamate dehydrogenase can be obtained by PCR (polymerase chain reaction; White, TJ et al., Trends Genet. 5, 185 (1989)) using primers designed based on the nucleotide sequence of SEQ ID NO: 1 . A gene encoding glutamate dehydrogenase from other organisms can also be obtained by PCR from libraries of chromosomal or genomic DNA using oligonucleotides as primers constructed on the basis of known sequences of a bacterial gene or a bacterium gene of a different kind, or by hybridization using an oligonucleotide designed as a probe based sequence. Chromosomal DNA can be obtained from a bacterium that serves as a DNA donor by the method of Saito and Miura (see N. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Experiment Manual for Biotechnology, edited by The Society for Biotechnology, Japan, p97-98, Baifukan Co., Ltd., 1992) and the like.

Recombinant DNA is then constructed by ligation of a gene amplified by PCR with a vector DNA capable of functioning in a host bacterium. Examples of vectors capable of functioning in a host bacterium include vectors autonomously replicating in a host bacterium. Examples of a vector autonomously replicating in Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, (pHSG and pACYC are available from Takara Bio Inc.), RSF1010 (Gene vol. 75 (2), p271-288, 1989) , pBR322, pMW219, pMW119 (pMW is available from Nippon Gene Co., Ltd.), pSTV28, and pSTV29 (Takara Bio Inc.). Phage vector DNA can also be used.

To ligate the gene into the aforementioned vector, the vector is treated with a restriction enzyme corresponding to the recognition site in the terminal region of the DNA fragment containing the gene. Ligation is usually carried out using a ligase, such as T ₄ - DNA ligase. Methods for preparing plasmid DNA, DNA restriction and ligation, transformation, selection of nucleotides as a primer, etc. may be conventional methods known to a person skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989) and the like.

Thus prepared recombinant DNA is introduced into the bacterium in accordance with the traditional method of transformation. Examples of the method include electroporation (Gliesche, C. G., Can. J. Microbiol., 43, 2, 197-201 (1997)). It is also possible to increase the DNA penetration by treating the recipient cells with calcium chloride as described for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970), and introducing DNA into a competent cell prepared from a cell in a proliferation step as described for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)).

An increase in the number of copies of the gene can also be achieved by introducing multiple copies of the gene encoding glutamate dehydrogenase into the chromosomal DNA of the bacterium. To introduce multiple copies of the gene into the bacterial chromosome, homologous recombination is performed using the target sequences present on the chromosome in multiple copies. Such sequences with multiple copies in the chromosomal DNA can be repeated DNA or inverted repeats at the ends of the transposed elements. On the other hand, as disclosed in JP 2-109985 A, multiple copies of a gene can be introduced into chromosomal DNA by inserting the gene into a transposon and transferring it so that multiple copies of the gene are integrated into the chromosomal DNA. The integration of these genes into the chromosome can be confirmed by Southern hybridization using a portion of the gene as a probe.

In addition, gene expression can be enhanced, as described in international application WO 00/18935, by replacing the expression control sequence, such as the promoter, of the gene on the chromosomal DNA or the gene on the plasmid with a stronger promoter, amplification of the expression enhancing regulator, or deletion, or weakening the regulator, weakening gene expression. Examples of known strong promoters include the lac promoter, trp promoter, trc promoter, tac promoter, P _R or P _L phage λ promoters and tet promoter.

On the other hand, the action of the promoter can be enhanced, for example, by introducing a nucleotide substitution into the promoter. Examples of a method for increasing promoter strength and examples of strong promoters are described by Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128) or the like. In addition, it is known that several nucleotides in the region between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately before the start codon, significantly affect translation efficiency. Therefore, you can modify this sequence.

In addition, to increase the activity of glutamate dehydrogenase, a mutation can be introduced into the gene, leading to an increase in enzymatic activity. Examples of such a mutation include a mutation in the promoter sequence to increase the level of transcription of the gene encoding glutamate dehydrogenase, and a mutation in the coding region of this gene to increase the specific activity of glutamate dehydrogenase.

Since L-glutamate is a precursor in the biosynthesis of L-glutamine, citrulline, L-arginine, ornithine and L-proline, an increase in the activity of glutamate dehydrogenase leads to an increase in the production of all these amino acids. Glutamate dehydrogenase activity can be determined, for example, as described by Meers J. et al., (J. Gen. Microb. 64, 187-94 (1970)).

L-amino acid producing bacterium

As a bacterium according to the present invention, modified in such a way that the expression of the gdhA gene is enhanced (in order to increase the activity of glutamate dehydrogenase), a bacterium capable of producing an L-amino acid belonging to the glutamate family can be used.

The bacterium of the present invention can be obtained by enhancing the expression of the gdhA gene in a bacterium already possessing the ability to produce L-amino acids belonging to the glutamate family. On the other hand, the bacterium of the present invention can be obtained by imparting a bacterium in which the expression of the gdhA gene is already enhanced to the ability to produce an L-amino acid belonging to the glutamate family.

L-glutamic acid producing bacterium

Examples of parent strains used to produce the L-glutamic acid producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain VL334 thrC ⁺ (European patent EP 1172433). Strain E. coli VL334 (VKPM B-1641) is an auxotroph of L-isoleucine and L-threonine with mutations in the thrC and ilvA genes (US patent 4278765). The natural allele of the thrC gene was transferred to this strain by the general transduction method using bacteriophage P1 grown on cells of the natural E. coli K12 strain (VKPM B-7). The result was a strain, auxotroph for L-isoleucine, VL334thrC ⁺ (VKPM B-8961), which is capable of producing L-glutamic acid.

Examples of parent strains used to produce L-glutamic acid producing bacteria according to the present invention include, but are not limited to, strains defective in the activity of α-ketoglutarate dehydrogenase, or strains in which the expression of one or more genes encoding enzymes is enhanced biosynthesis of L-glutamic acid. Examples of such enzymes include glutamate dehydrogenase (gdh), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydrate, acnA, acnu zarzudenosefruzidazufarzudenosephase (glutamine), phosphate synthase pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerometase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogen phosphate dehydrogenase (gapA) isofluosophosphate pfkB) and glucose phosphatisomerase (pgi).

Examples of strains modified in such a way that the expression of the citrate synthetase gene, phosphoenolpyruvate carboxylase gene and / or glutamate dehydrogenase gene is enhanced include those described in European applications EP1078989A, EP955368A and EP952221A.

Examples of parent strains for the production of L-glutamic acid producing bacteria according to the present study also include strains in which enzyme activity is reduced or absent that catalyzes the synthesis of compounds other than L-glutamic acid branching from the main pathway of L-glutamic acid biosynthesis. Examples of such enzymes include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase dehydrogenase (ilvI), ) Bacteria belonging to the genus Escherichia, lacking the activity of α-ketoglutarate dehydrogenase or having a decreased activity of α-ketoglutarate dehydrogenase and methods for their preparation are described in US patents 5378616 and 5573945. Specifically, examples of such strains include the following strains:

E.coli W3110sucA :: Km ^r

E. coli AJ12624 (PERM BP-3853)

E. coli AJ12628 (PERM BP-3854)

E. coli AJ12949 (PERM BP-4881)

E. coli W3110sucA :: Km ^r is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as the “sucA gene”) in E. coli strain W3110. In this strain, the activity of α-ketoglutarate dehydrogenase is completely absent.

Other examples of bacteria producing L-glutamic acid include bacteria belonging to the genus Escherichia and resistant to aspartic acid antimetabolites and deficient in the activity of α-ketoglutarate dehydrogenase, for example strain AJ13199 (FERM BP-5807) (US patent 5908768), or FERM P-12379, further having low L-glutamic acid cleavage activity (US Pat. No. 5,393,671); E. coli strain AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) and the like.

Examples of the L-glutamic acid producing bacterium include mutant strains belonging to the genus Pantoea that are devoid of α-ketoglutarate dehydrogenase activity or have reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Examples of such strains are Pantoea ananatis AJ13356 strain (US Pat. No. 6,331,419), Pantoea ananatis AJ13356 strain deposited at the National Institute of Biological Sciences and Human Technologies, Agency for Industrial Science and Technology, Department of International Trade and Industry (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry) (currently called the National Institute of Advanced Industrial Science and Technology, International Organizational Depository for Patenting Purposes, Central 6, 1-1, Higashi 1-Cho me, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan - National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) February 19, 1998 and received FERM P-16645. Then, the strain was internationally deposited in accordance with the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number FERM BP-6615. The Pantoea ananatis strain AJ13356 does not have α-KGDH activity as a result of the destruction of the αKGDH-E1 gene subunit (sucA). The above strain, when isolated, was identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans AJ13356 strain. However, it was later classified as Pantoea ananatis based on the 16S rRNA nucleotide sequence and other evidence. Although strain AJ13356 was deposited with Enterobacter agglomerans at the above depository, for the purposes of this description it will be referred to as Pantoea ananatis.

L-proline producing bacterium

Examples of L-proline producing bacteria used as the parent strain of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 702ilvA (VKPM B-8012) deficient in the ilvA gene and capable of producing L-proline (European patent EP 1172433). The bacterium of the present invention can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-proline. Preferably, examples of such genes for bacteria producing L-proline include the proB gene encoding glutamate kinase with desensitized feedback regulation of L-proline (German patent 3127361). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that secrete the L-amino acid from a bacterial cell. Examples of such genes are the b2682 and b2683 genes (ygaZH genes) (European Patent Application EP1239041A2).

Examples of bacteria belonging to the genus Escherichia and having the ability to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B-12404 (UK patent GB 2075056), VKPM B-8012 (RF patent application 2000124295), plasmid mutants described in German patent DE 3127361, plasmid mutants described in Bloom FR et al (The 15 ^th Miami winter symposium, 1983, p. 34), and the like.

L-arginine producing bacterium

Examples of parental strains used to produce the L-arginine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) (US patent application 2002 / 058315 A1) and its derivatives containing mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli 382 strain (VKPM B-7926) (European patent application EP1170358A1), arginine producer strain into which the argA gene encoding N -acetylglutamate synthetase (European patent application EP1170361 1), and the like.

Examples of parent strains used to produce the L-arginine producing bacterium of the present invention also include strains in which the expression of one or more genes encoding L-arginine biosynthesis enzymes is enhanced. Examples of L-arginine biosynthesis enzymes include N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetyl glutamate kinase (argB), acetylornithine transaminase (argD), ornithinecarbamoyl transferase (argF), argin acid, argin acid) and carbamoyl phosphate synthetase (carAB).

Citrulline Producing Bacteria

Examples of parental strains used to produce the citrulline producer bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as N-acetylglutamate synthase mutant strains E. coli 237 / pMADS11, 237 / pMADS12 and 237 / pMADS13 (RU2215783, EP1170361B1, US6790647B2).

Also, the citrulline producing bacterium can be easily obtained from any arginine producing bacterium, for example, from E. coli 382 strain (VKPM B-7926), by inactivation of arginine succinate synthase encoded by the argG gene.

The phrase "inactivation of arginine succinate synthase" means that the bacterium is modified so that the modified bacterium contains inactive arginine succinate synthase, or it may also mean that the bacterium is not capable of synthesizing arginine succinate synthase. Inactivation of arginine succinate synthase can be accomplished by inactivation of the argG gene.

The phrase "inactivation of the argG gene" means that the modified gene encodes a fully non-functional protein. It is also possible that the region of the modified DNA is not capable of naturally expressing the gene due to deletion of part of the gene or the entire gene, shift of the reading frame of the gene, insertion of the missense / nonsense mutation (s), or modification of regions adjacent to the gene, including sequences that control gene expression such as promoter, enhancer, attenuator, ribosome binding site, etc.

The presence or absence of the argG gene on the bacterial chromosome can be determined by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by determining the amount of RNA transcribed from the gene using various known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of protein encoded by the argG gene can be determined by known methods, including SDS-PAGE electrophoresis followed by immunoblotting (Western blotting), etc.

The expression of the argG gene can be attenuated by introducing a mutation into the gene on the chromosome so that the intracellular activity of the protein encoded by the gene is reduced compared to that in the unmodified strain. Such a gene mutation may be the replacement of one or more bases for amino acid substitution in the encoded protein (the Missense mutation), the introduction of a stop codon (the "nonsense" mutation), the deletion of one or more bases for reading frame shift, insertion of the resistance gene an antibiotic, or a deletion of a gene or part thereof (Qiu, Z. and Goodman, MF, J. Biol. Chem., 272, 8611-8617 (1997); Kwon, DH et al, J. Antimicrob. Chemother., 46, 793-796 (2000)). The expression of the argG gene can also be attenuated by modifying the expression of regulatory sequences such as a promoter, Shine-Dalgarno (SD) sequence, etc. (PCT application WO95 / 34672; Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed that encodes a mutant protein with reduced activity, and the bacterium for its modification is transformed with a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced by homologous recombination with the mutant gene, and the resulting strain is selected. Such gene substitution using homologous recombination can be carried out using a linear DNA method known as “Red-dependent integration” or “Red-system integration” (Datsenko, KA, Wanner, BL, Proc.Natl.Acad.Sci.USA , 97, 12, 6640-6645 (2000), PCT application WO 2005/010175) or by a method using a plasmid whose replication is temperature sensitive (US Patent 6303383 or Japanese Patent Application JP 05-007491 A). Further, the introduction of a site-specific mutation by replacing a gene using the aforementioned homologous recombination can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107) or by traditional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).

Ornithine-producing bacteria

The ornithine producing bacterium can be easily obtained from any arginine producing bacterium, for example, E. coli 382 strain (VKPM B-7926), by inactivating the ornithine carbamoyltransferase encoded by the argF and argI genes. Methods for inactivating ornithine carbamoyltransferase are described above.

L-threonine producing bacterium

Examples of the parent strain for producing the L-threonine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain TDH-6 / pVIC40 (VKPM B-3996) (US Pat. Nos. 5,175,107 and 5,705,737) , E. coli strain NRRL-21593 (US patent 5939307), E. coli strain FERM BP-3756 (US patent 5474918), E. coli strains FERM BP-3519 and FERM BP-3520 (US patent 5376538), strain E. coli MG442 (Gusyatiner et al., Genetics, 14, 947-956 (1978)), strains of E. coli VL643 and VL2055 (European patent application EP 1149911 A) and the like.

Strain TDH-6 is deficient in the thrC gene, is able to assimilate sucrose, and contains the ilvA gene with a leaky mutation. The specified strain contains a mutation in the rhtA gene, which causes resistance to high concentrations of threonine and homoserine. Strain B-3996 contains the plasmid pVIC40, which was obtained by introducing into the vector derived from the RSF1010 vector the thrA * BC operon including the thrA mutant gene encoding aspartokinase-homoserine dehydrogenase 1, which has a significantly reduced feedback sensitivity to threonine inhibition. Strain B-3996 was deposited on November 19, 1987 at the All-Union Scientific Center for Antibiotics (RF, 117105 Moscow, Nagatinskaya St., 3-A) with inventory number RIA 1867. The strain was also deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF , 117545 Moscow, 1st Dorozhniy proezd, 1) April 7, 1987 with inventory number B-3996.

The E. coli strain VKPM B-5318 (European application 0593792 B) can also be used as the parent strain for producing the L-threonine producing bacterium according to the present invention. Strain B-5318 is prototrophic with respect to isoleucine, and the temperature-sensitive C1 repressor of the phage λ and P _R promoter replaces the regulatory region in the threonine operon on the plasmid pVIC40. Strain VKPM B-5318 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF, 117545 Moscow, 1st Dorozhniy proezd, 1) May 3, 1990 with accession number B-5318.

Preferably, the bacterium of the present invention is further modified so as to have increased expression of one or more of the following genes:

- a mutant thrA gene encoding aspartokinase-homoserine dehydrogenase 1, resistant to threonine inhibition by feedback type;

- thrB gene encoding homoserine kinase;

- thrC gene encoding threonine synthase;

- rhtA gene, presumably encoding a transmembrane protein;

the asd gene encoding aspartate β-semialdehyde dehydrogenase, and

- aspC gene encoding aspartate aminotransferase (aspartate transaminase).

The nucleotide sequence of the thrA gene encoding aspartokinase homoserine dehydrogenase I from Escherichia coli is known (nucleotide numbers 337 to 2799 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrA gene is located on the chromosome of E. coli K-12 strain between the thrL and thrB genes. The nucleotide sequence of the thrB gene encoding homoserine kinase from Escherichia coli is known (nucleotide numbers 2801 to 3733 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrB gene is located on the chromosome of E. coli K-12 strain between the thrA and thrC genes. The nucleotide sequence of the thrC gene encoding threonine synthase from Escherichia coli is known (nucleotide numbers 3734 to 5020 in the sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrC gene is located on the chromosome of E. coli K-12 strain between the thrB gene and the open uaaX reading frame. All three of these genes function as one threonine operon. To enhance the expression of a threonine operon, it is desirable to remove an attenuator region from the operon that affects transcription (PCT application WO2005 / 049808, PCT application WO2003 / 097839).

The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to threonine inhibition by feedback type, as well as the thrB and thrC genes, can be obtained as a single operon from the well-known plasmid pVIC40, which is represented in the E. coli VKPM producer strain B-3996. Plasmid pVIC40 is described in detail in US Pat. No. 5,705,371.

The rhtA gene is located at the 18th minute of the E. coli chromosome near the glnHPQ operon, which encodes the components of the glutamine transport system, the rhtA gene is identical to ORF1 (ybiF gene, nucleotide numbers 764 to 1651 in sequence with accession number AAA218541 in the GenBank database, gi: 440181) located between the pexB and ompX genes. A region of DNA expressed to form the protein encoded by the ORF1 reading frame was named the rhtA gene (rht: resistance to homoserine and threonine). The rhtA23 mutation has also been shown to represent an A-to-G substitution at position -1 with respect to the start of the ATG codon (abstract of the 17 ^th International Congress of Biochemistry and Molecular Biology, abstract of the 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology , San Francisco, California August 24-29, 1997, abstract No. 457; European application EP 1013765 A).

The nucleotide sequence of the asd gene from E. coli is known (nucleotide numbers 3572511 to 3571408 in sequence with accession number NC_000913.1 in the GenBank database, gi: 16131307) and can be obtained using PCR (polymerase chain reaction; link to White, TJ et al., Trends Genet., 5, 185 (1989)) using primers synthesized based on the nucleotide sequence of the gene. Asd genes from other microorganisms can be obtained in a similar way.

Also, the nucleotide sequence of the aspC gene from E. coli is known (nucleotide numbers 983742 to 984932 in the sequence with accession number NC_000913.1 in the GenBank database, gi: 16128895) and can be obtained by PCR. AspC genes from other microorganisms can be obtained in a similar way.

L-valine producing bacterium

Examples of parent strains used to produce L-valine producing bacteria according to the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as H-81 (VKPM B-8066), NRRL B-12287 and NRRL B-12288 (U.S. Patent No. 4,391,907). VKPM B-4411 (US Patent No. 5658766), VKPM B-7707 (European Patent Application EP1016710A2), and the like.

Examples of parental strains used to produce the L-valine producing bacterium of the present invention include, but are not limited to, strains modified to overexpress ilvGMEDA operon (US Pat. No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon that is necessary to attenuate expression so that the expression of the operon is not attenuated by the resulting L-valine. Further, it is desirable to destroy the ilvA gene in the operon in order to reduce the activity of threonine deaminase.

Examples of parental strains used to produce L-valine producing bacteria according to the present invention also include mutant strains having a mutation of the aminoacyl tRNA synthetase (US Pat. No. 5,658,766). For example, E. coli strain VL1970, which has a mutation in the ileS gene encoding isoleucine-tRNA synthetase, can be used. The strain E.coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on June 24, 1988 with accession number VKPM B-4411.

Further, mutant strains that require lipoic acid and / or with an insufficient amount of H ⁺ -ATPase (PCT application WO96 / 06926) can also be used as parent strains.

2. The method according to the present invention.

The method according to the present invention is a method for producing an L-amino acid, comprising the steps of growing a bacterium according to the present invention in a nutrient medium for the purpose of producing and accumulating an L-amino acid in a nutrient medium and isolating the L-amino acid from the culture fluid.

According to the present invention, the cultivation, isolation and purification of an L-amino acid from a culture or similar liquid may be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using a bacterium.

The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Ethanol is used as a carbon source. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. As vitamins, thiamine, yeast extract, and the like can be used.

The cultivation is preferably carried out under aerobic conditions, such as mixing the culture fluid on a rocking chair, shaking with aeration, at a temperature in the range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture medium.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then the L-amino acid can be isolated and purified by ion exchange chromatography, concentration and / or crystallization.

Brief Description of the Drawing

The drawing shows the construction of the plasmid pMgdh4.

Examples

The present invention will be described in more detail below with reference to the following non-limiting Examples.

Example 1. Construction of a strain with enhanced expression of the gdhA gene

At the first stage, the DNA fragment containing the gdhA gene was obtained by PCR using primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4) and E. coli chromosomal DNA MG1655 (ATCC 700926) as a template. The resulting PCR product of size ≈1.6 TPN purified on an agarose gel and then cloned at the BamHI and SalI sites in plasmid pMW119. Thus, the plasmid pMgdh4 was obtained (see drawing). To confirm the expression of the gdhA gene in a strain containing plasmid pMgdh4, competent cells of strain HB101 (ATCC 33694) were transformed with plasmid pMgdh4 and plasmid pMW119 (as a control). Glutamate dehydrogenase activity was determined in strains HB101 / pMW119 and HB101 / pMgdh4. The results are presented in Table 1. As follows from Table 1, the activity of glutamate dehydrogenase in strain HB101 / pMgdh4 was significantly higher than in strain HB101 / pMW119.

Example 2. The production of L-arginine strain E. coli 382 / pMgdh4

To analyze the effect of an increase in glutamate dehydrogenase activity on L-arginine production, the E. coli 382 L-arginine producing strain was transformed with the plasmid pMgdh4 to obtain strain 382 / pMgdh4. Strain 382 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on April 10, 2000 with accession number VKPM B-7926, then on May 18, 2001, this strain was internationally deposited according to terms of the Budapest Treaty.

Both strains, 382 and 382 / pMgdh4, were grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth, 0.3 ml of the resulting cultures were introduced into 2 ml of fermentation medium in 20 × 200 mm tubes, and cultures were grown at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of L-arginine accumulated in the medium was determined by paper chromatography, and the following composition of the mobile phase was used: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution of ninhydrin (2%) in acetone was used for visualization. A spot containing L-arginine was excised; L-arginine was eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-arginine was determined spectrophotometrically at a wavelength of 540 nm. The results of ten independent in vitro fermentations are presented in Table 2. As follows from Table 2, 382 / pMgdh4 accumulated more L-arginine than 382.

The composition of the used fermentation medium (g / l):

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate were sterilized separately. CaCO _{3 was} dry heat sterilized at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Example 3. The production of L-glutamic acid strain E. coli VL334thrC ⁺ / pMgdh4

To analyze the effect of an increase in the activity of glutamate dehydrogenase on the production of L-glutamic acid, the E. coli L-glutamic acid producing strain VL334thrC ⁺ (EP 1172433) can be transformed with plasmid pMgdh4 to obtain strain VL334thrC ⁺ / pMgdh4. Strain VL334thrC ^{+ was} deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) December 6, 2004 with accession number VKPM B-8961, then December 8, 2004, this strain was internationally deposited in accordance with the conditions Budapest Treaty.

Both strains of E. coli, VL334thrC + and VL334thrC ⁺ / pMgdh4, can be grown on L-agar plates at 37 ° C for 18-24 hours. Further, one loop of cells can be transferred to tubes containing 2 ml of fermentation medium. The fermentation medium contains glucose - 60 g / l, ammonium sulfate - 25 g / l, KH ₂ PO ₄ - 2 g / l, MgSO ₄ - 1 g / l, thiamine - 0.1 mg / ml, L-isoleucine - 70 μg / ml and chalk - 25 g / l, the pH is adjusted to 7.2. Glucose and chalk are sterilized separately. Cultivation can be carried out at 30 ° C for 3 days with stirring. After growing, the amount of L-glutamic acid obtained can be determined using paper chromatography (mobile phase composition: butanol-acetic acid-water = 4: 1: 1), followed by staining with ninhydrin (1% solution in acetone) and further eluting the obtained compounds in 50% ethanol with 0.5% CdCl ₂ .

Example 4. Production of L-Proline by E. coli strain 702ilvA / pMgdh4

To assess the effect of an increase in glutamate dehydrogenase activity on L-proline production, the E. coli 702ilvA L-proline producing strain can be transformed with plasmid pMgdh4c to obtain strain 702ilvA / pMgdh4. Strain 702ilvA was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Road passage, 1) with accession number VKPM B-8012, then on May 18, 2001, this strain was internationally deposited in accordance with the terms of the Budapest Treaty.

Both strains of E. coli, 702ilvA and 702ilvA / pMgdh4, can be grown for 18-24 hours at 37 ° C on plates with L-agar. Then fermentation using these strains can be carried out under the same conditions as described in Example 3.

Example 5. The production of citrulline strain E. coli 382ΔargG / pMgdh4

To assess the effect of an increase in glutamate dehydrogenase activity on citrulline production, the E. coli 382ΔargG citrulline producing strain can be transformed with plasmid pMgdh4 to obtain strain 382ΔargG / pMgdh4. The 382ΔargG strain can be obtained by deletion of the argG gene on the chromosome of strain 382 (VKPM B-7926) using the method proposed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), called "Red-dependent Integration". In accordance with this procedure, PCR primers homologous to both the region adjacent to the argG gene and the gene responsible for antibiotic resistance can be designed. Plasmid pMW118-attL-Cm-attR (WO 05/010175) can be used as a template in PCR.

Both strains of E. coli, 382ΔargG and 382ΔargG / pMgdh4, can be grown with stirring at 37 ° C for 18 hours in 3 ml of culture medium, and 0.3 ml of the resulting cultures can be transferred to 2 ml of fermentation medium in 20 × 200- tubes mm and can be cultured at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of citrulline obtained can be determined by paper chromatography using a mobile phase of the following composition: butanol-acetic acid-water = 4: 1: 1. Subsequent staining can be performed with ninhydrin (2% solution in acetone). A stain containing citrulline can be excised, citrulline can be eluted using a 0.5% aqueous solution of CdCl ₂ , and the amount of citrulline can be determined spectrophotometrically at a wavelength of 540 nm.

Possible composition of the fermentation medium (g / l):

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH is adjusted to 7.0.

Example 6. Production of ornithine by E. coli strain 382ΔargFΔargI / pMgdh4

To assess the effect of an increase in glutamate dehydrogenase activity on the production of ornithine, the E. coli 382ΔargFΔargI ornithine producing strain can be transformed with the plasmid pMgdh4 to obtain the 382ΔargFΔargI / pMgdh4 strain. Strain 382ΔargFΔargI can be obtained by successive deletions of the argF and argI genes on the chromosome of strain 382 (VKPM B-7926) using the method proposed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), called "Red-dependent Integration". In accordance with this procedure, two pairs of PCR primers can be constructed that are homologous to both the regions adjacent to the argF and argI genes and the gene responsible for antibiotic resistance. Plasmid pMW118-attL-Cm-attR (WO 05/010175) can be used as a template in PCR.

Both strains of E. coli, 382ΔargFΔargI and 382ΔargFΔargI / pMgdh4, can be grown with stirring at 37 ° C for 18 hours in 3 ml of culture medium, and 0.3 ml of the resulting cultures can be transferred to 2 ml of fermentation medium in 20 × 200- tubes mm and can be cultured at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of ornithine obtained can be determined by paper chromatography using a mobile phase of the following composition: butanol-acetic acid-water = 4: 1: 1. Subsequent staining can be performed with ninhydrin (2% solution in acetone). A stain containing citrulline can be excised, citrulline can be eluted using a 0.5% aqueous solution of CdCl ₂ , and the amount of citrulline can be determined spectrophotometrically at a wavelength of 540 nm.

Possible composition of the fermentation medium (g / l):

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 L-arginine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH is adjusted to 7.0.

Example 7. The production of L-threonine strain E. coli 3996 / pMgdh4

To assess the effect of an increase in glutamate dehydrogenase activity on L-threonine production, the E. coli B-3996 L-threonine producing strain can be transformed with plasmid pMgdh4 to obtain strain B-3996 / pMgdh4. Strain B-3996 was deposited on November 19, 1987 at the All-Union Scientific Center for Antibiotics (RF, 117105 Moscow, Nagatinskaya St., 3-A) with inventory number RIA 1867. This strain was also deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF , 117545 Moscow, 1st Dorozhniy proezd, 1) April 7, 1987 with inventory number B-3996.

Both strains of E. coli, B-3996 and B-3996 / pMgdh4, can be grown for 18-24 hours at 37 ° C on plates with L-agar. To obtain a seed culture, these strains can be grown at 32 ° C for 18 hours on a rotary shaker (250 rpm) in 20 × 200 mm test tubes containing 2 ml of L-broth with 4% sucrose. Then, 0.21 ml (10%) of the seed culture can be added to the fermentation medium. Fermentation can be carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells can be grown for 65 hours at 32 ° C with stirring (250 rpm).

After growing, the amount of L-threonine accumulated in the medium can be determined by paper chromatography using the mobile phase of the following composition: butanol: acetic acid: water = 4: 1: 1 (v / v). For visualization, a solution (2%) of ninhydrin in acetone can be used. A stain containing L-threonine can be excised; L-threonine can be eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-threonine can be estimated spectrophotometrically at a wavelength of 540 nm.

The composition of the fermentation medium (g / l):

Glucose 80.0 (NH ₄ ) ₂ SO ₄ 22.0 NaCl 0.8 KH ₂ PO ₄ 2.0 MgSO _{4 ·} 7H ₂ O 0.8 FeSO ₄ · 7H ₂ O 0.02 MnSO ₄ · 5H ₂ O 0.02 Thiamine hydrochloride 0.0002 Yeast extract 1.0 CaCO ₃ 30.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Example 8. The production of L-valine strain E. coli H-81 / pMgdh4

To analyze the effect of an increase in the activity of glutamate dehydrogenase on the production of L-valine, the producer strain L-valine H-81 was first obtained and can be transformed with plasmid pMgdh4.

Strains H-81 and H-81 / pMgdh4 can be cultured at 37 ° C for 18 hours in nutrient broth, and 0.1 ml of each of the obtained cultures can be inoculated in 2 ml of fermentation medium in 20 × 200 mm tubes and can be carried out cultivation at 32 ° C for 72 hours on a rotary shaker. After culturing for 48 hours and 72 hours, the amount of accumulated L-valine can be determined by TLC. For this purpose, 10 × 15 cm TLC plates coated with a 0.11 mm layer of Sorbfil silica gel without a fluorescent indicator can be used (Sorbpolymer Joint-Stock Company, Krasnodar, Russia). Sorbfil plates can be exposed in the mobile phase of the following composition: propan-2-ol: ethyl acetate: 25% aqueous ammonia: water = 40: 40: 7: 16 (v / v). A solution (2%) of ninhydrin in acetone can be used for visualization.

The composition of the fermentation medium (g / l):

Glucose 48 (NH ₄ ) ₂ SO ₄ fifteen KH ₂ PO ₄ 1.5 MgSO ₄ · 7H ₂ O one Mameno (total N) 0.4 CaCO ₃ 25

CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Although the invention has been described in detail with reference to the best mode of carrying out the invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention.

Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Table 1. Strain Glutamate dehydrogenase, nmol / min · mg protein HB101 / pMW119 399 HB101 / pMgdh4 3971

Table 2. Strain OD ₅₄₀ Arg, g / l 382LW 15.3 ± 0.6 11.0 ± 1.1 382LW / pMW-gdh4 15.3 ± 0.7 15.5 ± 0.6

Claims (6)

1. A bacterium belonging to the genus Escherichia is a producer of an L-amino acid belonging to the glutamate family, or L-valine, modified in such a way that the activity of glutamate dehydrogenase in this bacterium is increased. 2. The bacterium according to claim 1, characterized in that said activity is increased by enhancing the expression of the gdhA gene. 3. The bacterium according to claim 2, characterized in that the expression of the specified gdhA gene is enhanced by increasing the number of copies of the gene or modifying the sequence that controls the expression of the gene, leading to increased expression of the specified gene. 4. L-amino acid producing bacterium according to any one of claims 1 to 3, characterized in that said L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-arginine, ornithine and citrulline. 5. A method for producing an L-amino acid belonging to the glutamate family, or L-valine, comprising:
growing bacteria according to claim 1 in a nutrient medium and
the allocation of the specified L-amino acids from the culture fluid. 6. The method according to claim 5, characterized in that said L-amino acid belonging to the glutamate family is selected from the group consisting of L-glutamic acid, L-glutamine, L-proline, L-arginine, ornithine and citrulline.

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