Patent Description:
Methods for fermentative production of amino acids and peptides utilizing mutant microorganisms, or microorganisms resistant to various drugs, have been previously reported. Conventional methods for producing such mutant strains include subjecting microorganisms to a mutagenesis treatment such as UV-irradiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), followed by selecting the desired strain by using a suitable selection medium. Alternatively, mutant strains can be bred by the use of genetic engineering techniques, including overexpressing genes involved in the biosynthesis pathway of the desired amino acid and peptides.

Production of a tripeptide such as γ-Glu-Val-Gly has been achieved previously via chemical and enzymatic methods, but not using fermentation of microorganisms. For example, <CIT> describes a multi-step method that uses various synthase enzymes to produce γ-Glu-Val and then γ-Glu-Val-Gly. <CIT> describes methods of producing γ-Glu-Val-Gly crystals via basic enzymatic and chemical methods. However, the use of fermentation of microorganisms engineered to produce γ-Glu-Val-Gly has not been previously described.

γ-Glu-Val-Gly is known to be useful in both the food and fragrance industries. For example, the tripeptide has been reported to have superior kokumi qualities, and hence has been used to flavor tea (<CIT>), alcoholic beverages (<NPL>), condiments (<NPL>), and spices (<CIT>). Hence, efficient and successful overproduction of the tripeptide γ-Glu-Val-Gly by fermentation of microorganisms is highly desirable. <CIT> discloses enzymatic routes for producing γ-Glu-Val-Gly.

The effect of overexpression of the gene(s) encoding L-threonine <NUM>-dehydrogenase and/or <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase on production of tripeptide γ-Glu-Val-Gly by fermentation of a bacterium belonging to the genus Enterobacteriaceae has not been previously reported.

The present invention provides a method for producing γ-Glu-Val-Gly.

In the method as described herein, any γ-Glu-Val-Gly-producing bacterium that has been modified to overexpress both a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be used.

The bacterium to be modified (i.e. the bacterium before introducing the modification of overexpressing both the genes) is not particularly limited, so long as the bacterium can be modified to overexpress both a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity, and the bacterium thus modified (i.e. the bacterium after introducing the modification of overexpressing both the genes) is able to produce a tripeptide γ-Glu-Val-Gly. Examples of the bacterium are described hereinafter.

The phrase "a bacterium is able to produce a tripeptide γ-Glu-Val-Gly" used for a bacterium after introducing a modification can mean that the bacterium did not have an ability to produce a tripeptide γ-Glu-Val-Gly before introducing the modification, but has obtained the ability to produce a tripeptide γ-Glu-Val-Gly by introducing the modification. Also, the phrase "a bacterium is able to produce a tripeptide γ-Glu-Val-Gly" used for a bacterium after introducing a modification can mean that the bacterium is rendered able to produce a tripeptide γ-Glu-Val-Gly by introducing the modification.

The phrase "a γ-Glu-Val-Gly-producing bacterium" can be equivalent to the phrase "a bacterium that is able to produce a tripeptide γ-Glu-Val-Gly" or the phrase "a bacterium having an ability to produce a tripeptide γ-Glu-Val-Gly". The phrase "a γ-Glu-Val-Gly-producing bacterium" can mean a bacterium that is able to produce a tripeptide γ-Glu-Val-Gly by fermentation of the bacterium in a culture medium. The phrase "a γ-Glu-Val-Gly-producing bacterium" can also mean a bacterium that is able to produce, excrete or secrete, and/or cause accumulation of the tripeptide γ-Glu-Val-Gly in a culture medium and/or the bacterial cells when the bacterium is cultivated in the medium. The phrase "a γ-Glu-Val-Gly-producing bacterium" can specifically mean a bacterium that is able to produce, excrete or secrete, and/or cause accumulation of the tripeptide γ-Glu-Val-Gly in a culture medium and/or the bacterial cells to such a level that the tripeptide γ-Glu-Val-Gly can be collected from the culture medium and/or the bacterial cells when the bacterium is cultivated in the medium. The phrase "a bacterium is cultivated in a medium" can be equivalent to the phrase "a bacterium is cultured in a medium", and these phrases are known to persons of ordinary skill in the art. The phrase "a γ-Glu-Val-Gly-producing bacterium" can also specifically mean a bacterium that is able to produce, excrete or secrete, and/or cause accumulation of the tripeptide γ-Glu-Val-Gly in a culture medium in an amount larger than a non-modified strain, for example, a wild-type or parental strain such as Escherichia coli (E. coli) K-<NUM> including E. coli K-<NUM> MG1655. The phrase "a γ-Glu-Val-Gly-producing bacterium" can also specifically mean a bacterium that is able to produce and cause accumulation in the medium and/or the bacterial cells of an amount not less than <NUM>/L of the tripeptide γ-Glu-Val-Gly, for example, not less than <NUM>/L, or not less than <NUM>/L, or not less than <NUM>/L of the tripeptide γ-Glu-Val-Gly. The phrase "a γ-Glu-Val-Gly-producing bacterium" can particularly mean a bacterium that is able to produce and cause accumulation in the medium of an amount not less than <NUM>/L of the tripeptide γ-Glu-Val-Gly, for example, not less than <NUM>/L, or not less than <NUM>/L, or not less than <NUM>/L of the tripeptide γ-Glu-Val-Gly.

The phrase "a tripeptide γ-Glu-Val-Gly", which can be used interchangeably or equivalently to the phrase "y-Glu-Val-Gly" (also abbreviated as "y-EVG"), can mean a tripeptide, which is a peptide containing three amino acid residues covalently bonded to one another in a chain configuration, wherein the tripeptide as described herein contains the glycine (Gly) residue, the amino group of which is bonded to the carboxylic group of the valine (Val) residue, the amino group of which is bound to the carboxylic group at the γ-carbon atom of the glutamic acid (Glu) residue (PubChem CID: <NUM>).

The bacterium can produce a tripeptide γ-Glu-Val-Gly in a free form, or a salt or hydrate thereof, or an adduct thereof (e.g. an adduct formed by the γ-Glu-Val-Gly and another organic or inorganic compound), or a mixture of these. Therefore, the phrase "y-Glu-Val-Gly" can include not only a tripeptide γ-Glu-Val-Gly in a free form, but may also include a salt or hydrate of the γ-Glu-Val-Gly, or an adduct thereof (e.g. an adduct formed by the γ-Glu-Val-Gly and another organic or inorganic compound). That is, the phrase "y-Glu-Val-Gly" can mean a tripeptide γ-Glu-Val-Gly in a free form, a salt or hydrate thereof, an adduct thereof, or a mixture thereof. The phrase "y-Glu-Val-Gly" can particularly mean a tripeptide γ-Glu-Val-Gly in a free form, a salt thereof, or a mixture thereof. It is also acceptable that the bacterium can produce a tripeptide γ-Glu-Val-Gly either alone or as a mixture of the γ-Glu-Val-Gly and one or more kinds of other amino acids or peptides. The phrase "L-amino acid" can mean an amino acid in L-form (so-called L-enantiomer of an amino acid) such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. The phrase "peptide" can include, for example, a dipeptide such as, for example, γ-Glu-Val and Val-Gly.

The bacterium may inherently be able to produce a tripeptide γ-Glu-Val-Gly or may be modified to become able to produce a tripeptide γ-Glu-Val-Gly. Such modification can be attained by, for example, mutation or DNA recombination techniques. That is, the bacterium can be obtained by overexpressing a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity in a bacterium that inherently is able to produce a tripeptide γ-Glu-Val-Gly, or in a bacterium that has been modified to become able to produce a tripeptide γ-Glu-Val-Gly. Alternatively, the bacterium can be obtained by overexpressing a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity to render the bacterium able to produce a tripeptide γ-Glu-Val-Gly. That is, the bacterium can be modified to overexpress the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity so that the bacterium thus modified is able to produce a tripeptide γ-Glu-Val-Gly.

The bacterium as described herein can be, for example, a gram-negative bacterium, specific examples of which include a bacterium belonging to the family Enterobacteriaceae. The explanations given hereinafter to the bacterium can be applied mutatis mutandis to any bacterium that can be used equivalently in the method as described herein.

In the method as described herein, the bacteria belonging to the family Enterobacteriaceae can be from the genera Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, and so forth, and can be able to produce a tripeptide γ-Glu-Val-Gly. Specifically, those classified into the family Enterobacteriaceae according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (ncbi. gov/Taxonomy/Browser/wwwtax. cgi?id=<NUM>) can be used. Examples of bacterial strains from the family Enterobacteriaceae which can be modified include a bacterium of the genus Escherichia, Enterobacter or Pantoea.

Strains of Escherichia bacterium which can be modified to obtain Escherichia bacteria in accordance with the presently disclosed subject matter are not particularly limited, and specifically, can include those described in the work of Neidhardt et al. The species Escherichia coli (E. coli) is a particular example. Specific examples of E. coli include E. coli W3110 (ATCC <NUM>), E. coli MG1655 (ATCC <NUM>), and so forth, which are derived from the prototype wild-type strain, E. coli K-<NUM> strain. These strains are available from, for example, the American Type Culture Collection (ATCC) as explained above. Examples of the Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and so forth. Examples of the Pantoea bacteria include Pantoea ananatis (P. ananatis), and so forth. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of <NUM> rRNA, etc. A bacterium belonging to either genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into the family Enterobacteriaceae. ananatis strain is bred by genetic engineering techniques, P. ananatis AJ13355 strain (FERM BP-<NUM>), AJ13356 strain (FERM BP-<NUM>), AJ13601 strain (FERM BP-<NUM>) and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans. However, they were recently re-classified as P. ananatis on the basis of nucleotide sequencing of <NUM> rRNA and so forth as described above.

These strains are available from, for example, the American Type Culture Collection (ATCC; Address: P. Box <NUM>, Manassas, VA <NUM>, United States of America). That is, registration numbers are assigned to the respective strains, and the strains can be ordered by using these registration numbers (refer to atcc. The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.

The bacterium as described herein has been modified to overexpress a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity.

The phrase "a protein having L-threonine <NUM>-dehydrogenase activity" can mean a protein that causes catalysis of the following reaction: L-threonine + NAD+ <-> L-<NUM>-amino-<NUM>-oxobutanoate + NADH + <NUM>+ (Enzyme Commission (EC) number <NUM>. <NUM>; Boylan S. and Dekker E. , L-Threonine dehydrogenase. Purification and properties of the homogeneous enzyme from E. coli K-<NUM>,<NPL>). For example, a protein having L-threonine <NUM>-dehydrogenase activity can mean the protein having the amino acid sequence shown in SEQ ID NO: <NUM> and homologues thereof that can cause catalysis of the reaction of the NAD+-dependent oxidation of L-threonine to L-<NUM>-amino-<NUM>-oxobutanoate. The activity of a protein having L-threonine <NUM>-dehydrogenase activity can be determined by evaluating colorimetrically the formation of aminoacetone from L-threonine or monitoring the formation of NADH using a spectrophotometer (see Boylan S. and Dekker E. , <NUM>, and references therein). The phrase "a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity" can mean a protein that causes catalysis of the following reaction: glycine + acetyl-coenzyme A <-> L-<NUM>-amino-<NUM>-oxobutanoate + coenzyme A + H+ (EC <NUM>. <NUM>; acetyl-coenzyme A is also referred to as Ac-CoA; Mukherjee J. and Dekker E. , Purification, properties, and N-terminal amino acid sequence of homogeneous E. coli <NUM>-amino-<NUM>-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme, <NPL>). For example, a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can mean the protein having the amino acid sequence shown in SEQ ID NO: <NUM> and homologues thereof that can cause catalysis of the reaction of the cleavage of <NUM>-amino-<NUM>-oxobutanoate to glycine and acetyl-coenzyme A. The activity of a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be determined by evaluating colorimetrically the formation of aminoacetone from glycine and Ac-CoA or observing the condensation reaction at <NUM> between coenzyme A (also referred to as CoA) and <NUM>,<NUM>'-dithiobis-(<NUM>-nitrobenzoic acid) (see, for example, Mukherjee J. and Dekker E. , <NUM>, and references therein).

The protein concentration can be determined by the Bradford protein assay or the method of Lowry using bovine serum albumin (BSA) as a standard and a Coomassie dye (<NPL>; <NPL>).

An example of the protein having L-threonine <NUM>-dehydrogenase activity can include the protein having the amino acid sequence shown in SEQ ID NO: <NUM>. The amino acid sequence shown in SEQ ID NO: <NUM> can be encoded by the nucleotide sequence shown in SEQ ID NO: <NUM>, which corresponds to the tdh gene. That is, an example of the protein having L-threonine <NUM>-dehydrogenase activity can include a tdh gene. The tdh gene of E. coli encodes the L-threonine <NUM>-dehydrogenase TDH, NAD(P)-binding (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. b3616; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P07913). The tdh gene (GenBank, accession No. NC_000913. <NUM>; nucleotide positions: <NUM> to <NUM>, complement; Gene ID: <NUM>) is located between the kbl gene and the waaH gene on the same strand of the chromosome of E. coli strain K-<NUM>. The nucleotide sequence of the tdh gene (SEQ ID NO: <NUM>) and the amino acid sequence of the TDH protein (SEQ ID NO: <NUM>) encoded by the tdh gene of E. coli are known. Moreover, homologues of TDH from different bacterial species are also known such as, for example, the homologues native to the bacteria belonging to the family Enterobacteriaceae, including the species E. coli having the TDH of SEQ ID NO: <NUM> (identity: <NUM>%), Shigella flexneri (identity: <NUM>%), Salmonella enteric (identity: <NUM>%), Klebsiella pneumonia (identity: <NUM>%), Enterobacter cloacae (identity: <NUM>%), P. ananatis (identity: <NUM>%); the family Burkholderiaceae, including the species Burkholderia mallei (identity: <NUM>%), Paraburkholderia xenovorans (identity: <NUM>%); the family Rhizobiaceae, including the species Rhizobium etli (identity: <NUM>%); the family Xanthomonadaceae, including the species Xanthomonas axonopodis (identity: <NUM>%); and so forth (see, for example, the NCBI database, National Center for Biotechnology Information, http://www. gov/protein/). Therefore, examples of the proteins having L-threonine <NUM>-dehydrogenase activity can also include the proteins that are homologues of the protein having the amino acid sequence shown in SEQ ID NO: <NUM>.

An example of the protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be the protein having the amino acid sequence shown in SEQ ID NO: <NUM>. The amino acid sequence shown in SEQ ID NO: <NUM> can be encoded by the nucleotide sequence shown in SEQ ID NO: <NUM> which corresponds to the kbl gene. That is, an example of the protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can include a kbl gene. The kbl gene of E. coli encodes the <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase KBL (synonyms: <NUM>-amino-<NUM>-ketobutyrate coenzyme A ligase, <NUM>-amino-<NUM>-oxobutanoate glycine-lyase (CoA-acetylating), glycine C-acetyltransferase, aminoacetone synthetase, aminoacetone synthase) (KEGG, entry No. b3617; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P0AB77). The kbl gene (GenBank, accession No. NC_000913. <NUM>; nucleotide positions: <NUM> to <NUM>, complement; Gene ID: <NUM>) is located between the yibB gene and the tdh gene on the same strand of the chromosome of E. coli strain K-<NUM>. The nucleotide sequence of the kbl gene (SEQ ID NO: <NUM>) and the amino acid sequence of the KBL protein (SEQ ID NO: <NUM>) encoded by the kbl gene of E. coli are known. Moreover, homologues of KBL from different bacterial species are also known such as, for example, the homologues native to the bacteria belonging to the family Enterobacteriaceae, including the species E. coli having the KBL of SEQ ID NO: <NUM> (identity: <NUM>%), Shigella dysenteriae (identity: <NUM>%), Citrobacter farmer (identity: <NUM>%), Salmonella enterica (identity: <NUM>%), P. ananatis (identity: <NUM>%); the family Yersiniaceae, including the species Serratia marcescens (identity: <NUM>%); from the family Morganellaceae, including species Xenorhabdus khoisanae (identity: <NUM>%); from the family Erviniaceae, including the species Erwinia sp. <NUM> (identity: <NUM>%); from the family Xanthomonadaceae, including the species Lysobacter spongiicola (identity: <NUM>%), Stenotrophomonas maltophilia (identity: <NUM>%); and so forth (see, for example, the NCBI database). Therefore, examples of the proteins having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can also be the proteins that are homologues of the protein having the amino acid sequence shown in SEQ ID NO: <NUM>.

The phrase "a bacterium has been modified to overexpress a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity" can mean that the bacterium has been modified in such a way that in the modified bacterium the total activity of the corresponding gene product that causes catalysis of the reaction of the NAD+-dependent oxidation of L-threonine to L-<NUM>-amino-<NUM>-oxobutanoate and the total activity of the corresponding gene product that causes catalysis of the reaction of the cleavage of <NUM>-amino-<NUM>-oxobutanoate to glycine and acetyl-coenzyme A are both increased, or the expression level (i.e. expression amount) of the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the expression level (i.e. expression amount) of the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity are both increased, as compared with a non-modified strain. The phrase "a non-modified strain" can refer to a bacterial strain that can serve as a reference for the above comparison. The phrase "a non-modified strain" is also referred to as "a non-modified bacterium" or "a non-modified bacterial strain". Examples of the non-modified strain can include a wild-type or parental strain of a bacterium belonging to the family Enterobacteriaceae such as a bacterium belonging to the genus Escherichia or Pantoea including E. coli and P. Specific examples of the non-modified strain can include the strains E. coli MG1655 (ATCC <NUM>) and W3110 (ATCC <NUM>) and the strain P. ananatis AJ13355 (FERM BP-<NUM>).

The bacterium modified to overexpress a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be a bacterium in which the total activity of the corresponding gene product that causes catalysis of the reaction of the NAD+-dependent oxidation of L-threonine to L-<NUM>-amino-<NUM>-oxobutanoate and the total activity of the corresponding gene product that causes catalysis of the reaction of the cleavage of <NUM>-amino-<NUM>-oxobutanoate to glycine and acetyl-coenzyme A can be both increased by, for example, increasing (i.e. enhancing) the expression level of the gene encoding the protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding the protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity, or increasing the activity per molecule (may be referred to as a specific activity) of the proteins encoded by said genes, as compared with a non-modified strain, for example, a wild-type or parental strain. An increase in total activity of a protein can be measured as, for example, an increase in the activity of the protein per cell, which may be an average activity of the protein per cell. The bacterium may be modified so that the activity of the protein having L-threonine <NUM>-dehydrogenase activity per cell and/or the activity of the protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity per cell are/is increased to, for example, <NUM>% or more, <NUM>% or more, <NUM>% or more, of the activity of that protein(s) in a non-modified strain.

The phrase "a bacterium has been modified to overexpress a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity" can also mean that the bacterium has been modified in such a way that in the modified bacterium the expression level (i.e. expression amount) of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the expression level (i.e. expression amount) of a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity are both increased as compared with a non-modified strain, for example, a wild-type or parental strain. Therefore, the phrase "a gene is overexpressed" can be equivalent to the phrase "the expression of a gene is enhanced or increased" or the phrase "the expression level of a gene is enhanced or increased". An increase in the expression level of a gene can be measured as, for example, an increase in the expression level of the gene per cell, which may be an average expression level of the gene per cell. The bacterium may be modified so that the expression level of the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity per cell and/or the expression level of the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity per cell are/is increased to, for example, <NUM>% or more, <NUM>% or more, <NUM>% or more, of the expression level of that gene(s) in a non-modified strain.

The aforementioned descriptions concerning overexpression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can also be independently applied to each of these genes.

Methods for modifying a bacterium to overexpress both a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity, and methods which can be used to enhance expression of the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity in the bacterium as described herein may depend on the bacterium that is chosen for the modification. Any method for gene overexpression may be used, so long as the overexpression of the gene can be attained using that method. Therefore, the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be overexpressed using one method for gene overexpression, or the genes can be overexpressed using different methods for gene overexpression.

Methods which can be used to enhance expression of a gene can include, but are not limited to, increasing the copy number of the gene, such as the copy number of the gene in the chromosome of the bacterium and/or in the autonomously replicating plasmid harbored by the bacterium. The copy number of a gene can be increased by, for example, introducing the gene into the chromosome of the bacterium and/or introducing an autonomously replicating vector containing the gene into the bacterium. Such increasing of the copy number of a gene can be carried out according to genetic engineering methods known to the one of ordinary skill in the art.

Examples of the vectors can include, but are not limited to, broad-host-range plasmids such as pMW <NUM>/<NUM>, pBR322, pUC19, and the like. Multiple copies of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and/or a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can also be introduced into the chromosomal DNA of a bacterium by, for example, homologous recombination, Mu-driven integration, or the like. Only one copy, or two or more copies of each gene may be introduced. For example, homologous recombination can be carried out using a sequence that is present in multiple copies in the chromosomal DNA as a target to introduce multiple copies of a gene into the chromosomal DNA. Sequences with multiple copies in the chromosomal DNA can include, but are not limited to, repetitive DNA or inverted repeats present at the end of a transposable element. In addition, it is possible to incorporate a gene into a transposon and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA. By using Mu-driven integration, more than <NUM> copies of the gene can be introduced into the chromosomal DNA during a single act (<NPL>).

The bacterium as described herein can be modified to overexpress a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity so that the genes are present in the bacterium after introduction of the genes. Also, the bacterium can be modified in such a way that the activity of the protein having L-threonine <NUM>-dehydrogenase activity and the activity of the protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be determined in the modified bacterium. That is, any bacterium that does not natively or naturally have a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be used, so long as the bacterium can be modified to overexpress the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity so that the activity of the protein having L-threonine <NUM>-dehydrogenase activity and the activity of the protein having the activity of <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase can be determined in the modified bacterium and the modified bacterium is able to produce a tripeptide γ-Glu-Val-Gly as described herein.

The bacterium as described herein has been modified to harbor a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity. The gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be overexpressed in the bacterium in such a way that the genes are present on different nucleic acid molecules. Alternatively, the genes can be introduced into the bacterium in such a way that the genes are present on one nucleic acid molecule. For example, the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having an activity of <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase may be present on one expression vector or on the chromosome. Alternatively, the genes may be present on two different expression vectors. Also, alternatively, a gene may be present on one expression vector and another gene may be present on the chromosome.

As a bacterium belonging to the family Enterobacteriaceae can be an example of the bacterium as described herein, the methods for overexpression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity in a bacterium belonging to the family Enterobacteriaceae are described hereinafter.

A method for the overexpression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity in an Enterobacteriaceae bacterium can be introducing a nucleic acid (DNA) having the gene(s) into the Enterobacteriaceae bacterium. Methods for introducing a nucleic acid such as, for example, a gene, a vector, and the like, into an Enterobacteriaceae bacterium can include, but are not limited to, genetic engineering methods known to the person of ordinary skill in the art, and these are not particularly limited. In the bacterium as described herein, the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be present on a vector that autonomously replicates outside of the chromosome such as a plasmid, or may be incorporated into the chromosome. In addition, as described above, to construct the bacterium as described herein, introduction of the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity, and impartation or enhancement of the ability to produce a tripeptide γ-Glu-Val-Gly can be performed in any order.

The other methods which can be used to enhance expression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can include increasing the expression level of the genes by modification of expression regulatory region(s) of these genes. Expression regulatory region(s) of genes can be modified by, for example, replacing the native expression regulatory region(s) of these genes with native and/or modified foreign regulatory region(s). The phrase "an expression regulatory region" can also be referred to as "an expression regulatory sequence". When the genes encoding a protein having L-threonine <NUM>-dehydrogenase activity and/or a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity are organized in an operon structure, the method which can be used to enhance expression of the genes also can include increasing the expression level of the operon having these genes by modification of expression regulatory region(s) of the operon, wherein the modification can be carried out by, for example, replacing the native expression regulatory region(s) of the operon with native and/or modified foreign regulatory region(s). In this method, the expression of one or more genes in the operon, including the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and the gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity, can be enhanced at the same time.

Expression regulatory regions can be exemplified by promoters, enhancers, attenuators and termination signals, anti-termination signals, ribosome-binding sites (RBS) and other expression control elements, such as regions to which repressors or inducers bind and/or binding sites for transcriptional and translational regulatory proteins, for example, in the transcribed mRNA. Such regulatory regions are described, for example, in <NPL>). Modification of expression regulatory region(s) of a gene can be combined with increasing the copy number of the gene (see, for example, <NPL>; <NPL>).

The exemplary promoters suitable for enhancing expression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be the potent promoters that are stronger than the native promoters of the genes. For example, the lac promoter, the trp promoter, the trc promoter, the tac promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter (derived from the genus Bifidobacterium), and the PR and PL promoters of lambda phage are all known to be potent promoters. Potent promoters providing a high level of gene expression in a bacterium belonging to the family Enterobacteriaceae can be used. Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of a gene to obtain a stronger promoter function, thus resulting in the increased transcription level of the gene located downstream from the promoter. Furthermore, it is known that substitution of several nucleotides in the Shine-Dalgarno (SD) sequence, and/or in the spacer between the SD sequence and the start codon, and/or a sequence immediately upstream and/or downstream from the start codon in the ribosome-binding site greatly affects the translation efficiency of mRNA. For example, a <NUM>-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (<NPL>; <NPL>).

The copy number of a gene or the presence or absence of a gene can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like. The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), or mass spectrometry analysis of the protein samples, and the like.

Methods for manipulation with recombinant molecules of DNA and molecular cloning such as preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an oligonucleotide as a primer, incorporation of mutations, and the like may be ordinary methods well-known to the persons of ordinary skill in the art. These methods are described, for example, in <NPL>) or <NPL>); <NPL>).

Any methods for manipulation with recombinant DNA can be used including conventional methods such as, for example, transformation, transfection, infection, conjugation, and mobilization. Transformation, transfection, infection, conjugation or mobilization of a bacterium with the DNA encoding a protein can impart to the bacterium the ability to synthesize the protein encoded by the DNA. Methods of transformation, transfection, infection, conjugation, and mobilization include any known methods. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells of E. coli K-<NUM> to DNA has been reported for efficient DNA transformation and transfection (<NPL>). Methods of specialized and/or generalized transduction have been described (<NPL>;<NPL>). Other methods for random and/or targeted integration of DNA into the host microorganism can be applied, for example, "Mu-driven integration/amplification" (<NPL>), "Red/ET-driven integration" or "λRed/ET-mediated integration" (<NPL>; <NPL>). Moreover, to insert multiple desired genes in addition to Mu-driven replicative transposition (<NPL>), and chemically induce chromosomal evolution based on recA-dependent homologous recombination resulting in an amplification of the desired genes (<NPL>), methods can be used which utilize different combinations of transposition, site-specific and/or homologous Red/ET-mediated recombinations, and/or P1-mediated generalized transduction (see, for example, <NPL>; <NPL>).

Methods for overexpression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity in bacterial species other than a bacterium belonging to the family Enterobacteriaceae can be applied mutatis mutandis by referring to the methods described herein for the bacterium belonging to the family Enterobacteriaceae, or those methods can be used that are known to the persons of ordinary skill in the art. Furthermore, it is within the ordinary skill to use common methods that are suitable for gene overexpression in a bacterium belonging to the family Enterobacteriaceae. Moreover, the methods suitable for the gene overexpression in an Enterobacteriaceae bacterium can be appropriately modified and used to overexpress a gene in other species of bacteria, and contrariwise. Therefore, the methods for gene overexpression as described herein may, virtually, be applied to any bacterium as described herein.

Hereinafter, variants of the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and variants of the protein having L-threonine <NUM>-dehydrogenase activity, specifically variants of those native to E. coli, will be described. The below descriptions of such variants of the gene and protein can also be applied mutatis mutandis to any gene and protein, including a gene native to a bacterial species other than E. coli and encoding a protein having L-threonine <NUM>-dehydrogenase activity and the encoded protein, and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity and the encoded protein.

There may be differences in DNA sequences between the bacterial families, genera, species or strains. Therefore, the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity is not limited to the gene having the nucleotide sequence shown in SEQ ID NO: <NUM>, but may include genes each of which has a variant nucleotide sequence of SEQ ID NO: <NUM> and encodes a protein having L-threonine <NUM>-dehydrogenase activity. Similarly, the protein having L-threonine <NUM>-dehydrogenase activity is not limited to the protein having the amino acid sequence shown in SEQ ID NO: <NUM>, but may include proteins each of which has a variant amino acid sequence of SEQ ID NO: <NUM> and has L-threonine <NUM>-dehydrogenase activity. Examples of such variant nucleotide sequence or variant amino acid sequence may include homologues of and artificially modified ones of the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity exemplified above or of the protein having L-threonine <NUM>-dehydrogenase activity exemplified above.

The phrase "a variant protein" can mean a protein which has a variant amino acid sequence of SEQ ID NO: <NUM>.

The phrase "a variant protein" can specifically mean a protein which has one or more mutations in the sequence as compared with the amino acid sequence shown in SEQ ID NO: <NUM>, whether they are substitutions, deletions, insertions, and/or additions of one or several amino acid residues, but which still maintains the L-threonine <NUM>-dehydrogenase activity as described herein, or of which the three-dimensional structure is not significantly changed relative to the non-modified protein such as, for example, the protein having the amino acid sequence shown in SEQ ID NO: <NUM>. The number of changes in the variant protein depends on the position of amino acid residue(s) in the three-dimensional structure of the protein or the type of amino acid residue(s). It can be, but is not strictly limited to, <NUM> to <NUM>, in another example <NUM> to <NUM>, in another example <NUM> to <NUM>, in another example <NUM> to <NUM>, and in another example <NUM> to <NUM>, in SEQ ID NO: <NUM>. This is possible because amino acids can have high homology to one another, so that the activity of a protein is not affected by a change between such amino acids, or the three-dimensional structure of a protein is not significantly changed relative to the corresponding non-modified protein by a change between such amino acids. Therefore, the variant protein may be a protein having an amino acid sequence having a homology, defined as the parameter "identity" when using the computer program BLAST, of not less than <NUM>%, of not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, or not less than <NUM>% with respect to the entire amino acid sequence shown in SEQ ID NO: <NUM>, as long as the L-threonine <NUM>-dehydrogenase activity of the protein is maintained, or the three-dimensional structure of the protein is not significantly changed relative to the non-modified protein such as, for example, the protein having the amino acid sequence shown in SEQ ID NO: <NUM>.

The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation(s). The representative conservative mutation can be a conservative substitution. The conservative substitution can be, but is not limited to, a substitution, wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Ala, Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gln and Asn, if the substitution site is a polar amino acid; among Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having hydroxyl group. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln, substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu, substitution of Asn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, Ile or Leu for Val. The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can also be a non-conservative mutation(s) provided that the mutation(s) is/are compensated by one or more secondary mutation(s) in a different position(s) of amino acids sequence so that the L-threonine <NUM>-dehydrogenase activity of the variant protein is maintained, or the three-dimensional structure of the protein is not significantly changed relative to the non-modified protein such as, for example, the protein having the amino acid sequence shown in SEQ ID NO: <NUM>.

To evaluate the degree of protein or DNA homology, several calculation methods can be used, such as a BLAST search, FASTA search and ClustalW method. The BLAST (Basic Local Alignment Search Tool, www. gov/BLAST/) search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Karlin S. and Altschul S. ("<NPL>; "<NPL>). The computer program BLAST calculates three parameters: score, identity and similarity. The FASTA search method is described by <NPL>). The ClustalW method is described by<NPL>). In this specification, the phrase "homology" may mean "identity", which is the identity of amino acid sequences or nucleotide sequences. The sequence identity between two sequences is calculated as the ratio of residues matching in the two sequences when aligning the two sequences so as to achieve a maximum alignment with each other. The phrase "identity" between amino acid sequences may specifically mean an identity calculated by blastp with default scoring parameters (i.e. Matrix, BLOSUM62; Gap Costs, Existence = <NUM>, Extension = <NUM>; Compositional Adjustments, Conditional compositional score matrix adjustment), unless otherwise stated. The phrase "identity" between nucleotide sequences may specifically mean an identity calculated by blastn with default scoring parameters (i.e. Match/Mismatch Scores = <NUM>, -<NUM>; Gap Costs = Linear), unless otherwise stated.

The phrase "a variant nucleotide sequence" can mean a nucleotide sequence which encodes a protein having L-threonine <NUM>-dehydrogenase activity, such as the protein having the amino acid sequence shown in SEQ ID NO: <NUM>, using any synonymous amino acid codons according to the standard genetic code table (see, e.g., <NPL>). Therefore, the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity can be a gene having a variant nucleotide sequence due to the degeneracy of the genetic code.

The phrase "a variant nucleotide sequence" can also mean a nucleotide sequence that is able to hybridize under stringent conditions with a nucleotide sequence complementary to the sequence shown in SEQ ID NO: <NUM> or a probe that can be prepared from the nucleotide sequence provided that it encodes a protein having L-threonine <NUM>-dehydrogenase activity. The phrase "stringent conditions" can include conditions under which a specific hybrid, for example, a hybrid having homology, defined as the parameter "identity" when using the computer program BLAST, of not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, not less than <NUM>%, or not less than <NUM>%, is formed, and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed. For example, stringent conditions can be exemplified by washing one time or more, or in another example, two or three times, at a salt concentration of <NUM> x SSC (standard sodium citrate or standard sodium chloride), <NUM>% SDS (sodium dodecyl sulphate) at <NUM>, <NUM> x SSC, <NUM>% SDS at <NUM>, or <NUM> x SSC, <NUM>% SDS at <NUM>. The duration of washing can depend on the type of membrane used for the blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Amersham Hybond™-N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is <NUM> minutes. The washing step can be performed <NUM> to <NUM> times. As the probe, a part of the sequence complementary to the sequence shown in SEQ ID NO: <NUM> may also be used. Such a probe can be produced by PCR (polymerase chain reaction; refer to <NPL>) using oligonucleotides as primers prepared on the basis of the sequence shown in SEQ ID NO: <NUM> and a DNA fragment containing the nucleotide sequence to be used as the probe as a template. The length of the probe is recommended to be ><NUM> bp; it can be suitably selected depending on the hybridization conditions, and is usually <NUM> bp to <NUM> kbp. For example, when a DNA fragment having a length of about <NUM> bp is used as the probe, the washing conditions after the hybridization can be, for example, <NUM> x SSC, <NUM>% SDS at <NUM>, <NUM> or <NUM>.

The phrase "a variant nucleotide sequence" can also mean a nucleotide sequence that encodes a variant protein.

Since the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and native to E. coli has already been elucidated (see above), the gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and native to E. coli or a variant nucleotide sequence thereof can be obtained by cloning from E. coli by PCR utilizing DNA of E. coli and oligonucleotide primers prepared based on the nucleotide sequence of the tdh gene native to E. coli; or a mutagenesis method of treating a DNA containing the tdh gene, in vitro, for example, with hydroxylamine, or a mutagenesis method of treating E. coli harboring the tdh gene with ultraviolet (UV) irradiation or a mutating agent such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the such treatment; or chemical synthesis as a full-length gene structure. Genes encoding the proteins having L-threonine <NUM>-dehydrogenase activity native to other oraganism such as a bacterium of the family Enterobacteriaceae other than E. coli or a variant nucleotide sequence thereof can be obtained in a similar manner.

The phrase "non-modified", which can be equivalent to the phrase "native", "natural", or "wild-type", in reference to a gene (for example, "a non-modified gene") and a protein (for example, "a non-modified protein"), can mean, respectively, a native gene and a native protein that exist naturally in, are expressed naturally in, and/or are produced naturally by an organism, specifically a non-modified strain of a bacterium, for example, a wild-type strain of a bacterium of the family Enterobacteriaceae such as, for example, the E. coli MG1655 strain (ATCC <NUM>), the E. coli W3110 strain (ATCC <NUM>), the P. ananatis AJ13355 strain (FERM BP-<NUM>), and so forth. A non-modified gene can encode a non-modified protein.

The phrase "native to" in reference to a protein or a nucleic acid native to a particular species of organisms such as, for example, a bacterial species, can refer to a protein or a nucleic acid that is native to that species. That is, a protein or a nucleic acid native to a particular species can mean the protein or the nucleic acid, respectively, that exists naturally in that species. A protein or a nucleic acid native to a particular species can be isolated from that species and sequenced using means known to the one of ordinary skill in the art. Moreover, as the amino acid sequence or the nucleotide sequence of a protein or nucleic acid, respectively, isolated from a species in which the protein or nucleic acid exists, can easy be determined, the phrase "native to" in reference to a protein or a nucleic acid can also refer to a protein or a nucleic acid that can be obtained using any means, for example, using a genetic engineering technique, including recombinant DNA technology, or a chemical synthesis method, or the like, so long as the amino acid sequence of the protein or the nucleotide sequence of the nucleic acid thus obtained is identical to the amino acid sequence of the protein or the nucleotide sequence of the nucleic acid that exists naturally in the species. The phrase "a protein" can include, but are not limited to, peptides, oligopeptides, polypeptides, proteins, enzymes, and so forth. The phrase "a nucleic acid" can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and can specifically include, but are not limited to, regulatory sequences, including promoters, attenuators, terminators, and the like, genes, intergenic sequences, sequences encoding signal peptides, pro-moieties of proteins, artificial amino acid sequences, and so forth. Specific examples of amino acid sequences and nucleotide sequences, and homologues thereof native to various species are described herein. Specific examples of proteins native to E. coli include TDH and KBL having the amino acid sequences shown in SEQ ID NOs: <NUM> and <NUM>, respectively. Specific examples of genes native to E. coli include tdh and kbl genes having the nucleotide sequences shown in SEQ ID NOs: <NUM> and <NUM>, respectively.

There are other genes the expression of which can be altered by either down-regulating the expression (i.e. attenuating the expression) or overexpressing the gene, and such alteration(s) can have a positive effect on the production of the tripeptide γ-Glu-Val-Gly during cultivation of the bacterium as described herein. The bacterium may have been modified to have such alteration(s). The bacterium may have been specifically modified to have any one of or any combination of such alteration(s).

The phrase "a bacterium has been modified to attenuate the expression a gene" can mean that the bacterium has been modified in such a way that in the modified bacterium the total activity of the corresponding gene product is decreased, or the expression level (i.e. expression amount) of the gene is decreased, as compared with a non-modified strain. The bacterium may be modified so that the activity of a protein encoded by an objective gene per cell is decreased to, for example, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, or <NUM>%, of the activity of the protein in a non-modified strain. The bacterium may be modified so that the expression level of an objective gene per cell is decreased to, for example, <NUM>% or less, <NUM>% or less, <NUM>% or less, <NUM>% or less, or <NUM>%, of the expression level of the gene in a non-modified strain.

The aforementioned description concerning overexpression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity can be applied mutatis mutandis to other genes.

Specific methods for attenuating the expression of gene(s) or overexpressing gene(s) are well known to persons of ordinary skill in the art. Attenuation of the expression of a gene can be attained by, for example, disrupting or deleting the gene. Overexpression of a gene can be attained by, for example, a similar manner as that for overexpression of a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity.

Examples of such alterations can include attenuating the expression of genes encoding enzymes that prevent glycine degradation and enzymes that increase glutamic acid biosynthesis. These alterations can include attenuating the expression of a gcvP gene and attenuating the expression of sucAB operon genes. The gcvP gene encodes a component of the glycine cleavage system, which prevents glycine degradation due to a glycine decarboxylase reaction (<NPL>). The sucAB genes encode two subunits of <NUM>-ketoglutarate dehydrogenase (KGDH). Attenuation of the expression of the sucAB genes, such as decrease in the activity of proteins encoded by the genes, can result in a decrease in KGDH activity. For example, it has been reported that attenuation of the expression of the sucAB genes results in decreasing KGDH activity by <NUM>%, thereby elevating glutamic acid accumulation <NUM>-fold (<CIT>). A decrease in KGDH activity is useful for γ-Glu-Val-Gly production due to its positive influence on glutamic acid synthesis.

Examples of such alterations can also include overexpression of genes encoding proteins involved in the production of L-valine and proteins involved in export of the tripeptide γ-Glu-Val-Gly. These alterations can include overexpression of ilvGMEDA operon genes (<CIT>) and the tolC gene. The tolC gene encodes an outer membrane protein involved in a range of tripartite efflux complexes (<NPL>). The ilvGMEDA operon can include an ilvG gene having a mutation that results in regeneration of the activity of acetohydroxylic acid synthase II (AHAS II) encoded by the ilvG gene (<CIT>; <CIT>).

The bacterium can have, in addition to the properties already mentioned, other specific properties such as various nutrient requirements, drug resistance, drug sensitivity, and drug dependence.

The method of producing a tripeptide γ-Glu-Val-Gly using a bacterium as described herein includes the steps of cultivating (also called culturing) the bacterium in a culture medium to allow γ-Glu-Val-Gly to be produced, excreted or secreted, and/or accumulated in the culture medium or in the bacterial cells, or both, and collecting the γ-Glu-Val-Gly from the culture medium and/or the bacterial cells. The method may include, optionally, the step of purifying a target tripeptide γ-Glu-Val-Gly from the culture medium and/or the bacterial cells. The γ-Glu-Val-Gly can be produced in such a form as described above. The γ-Glu-Val-Gly can be produced particularly in a free form or as a salt thereof, or as a mixture of them. For example, sodium, potassium, ammonium, and the like salts can be produced by the method. This is possible as amino acids can react under fermentation conditions with each other or a neutralizing agent such as an inorganic or organic acidic or alkaline substance in a typical acid-base neutralization reaction to form a salt that is the chemical feature of amino acids which is apparent to persons of ordinary skill in the art.

The cultivation of the bacterium, and collection, and, optionally, purification of a tripeptide γ-Glu-Val-Gly from the medium and the like may be performed in a manner similar to the conventional fermentation methods wherein an L-amino acid is produced using a microorganism. The culture medium can be either a synthetic or natural medium such as a typical medium that contains a carbon source, a nitrogen source, a sulphur source, a phosphorus source, inorganic ions, and other organic and inorganic components as required. As the carbon source, saccharides such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches; alcohols such as ethanol, glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; fatty acids, and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as of soy bean hydrolysate; ammonia gas; aqueous ammonia; and the like can be used. Furthermore, peptone, yeast extract, meat extract, malt extract, corn steep liquor, and so forth can also be utilized. The medium may contain one or more types of these nitrogen sources. The sulphur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, and the like. The medium can contain a phosphorus source in addition to the carbon source, the nitrogen source and the sulphur source. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphate polymers such as pyrophosphoric acid and so forth can be utilized. Vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, vitamin B12, required substances, for example, organic nutrients such as nucleic acids such as adenine and RNA, amino acids, peptone, casamino acid, yeast extract, and the like may be present in appropriate, even if trace, amounts. Other than these, small amounts of calcium phosphate, iron ions, manganese ions, and so forth may be added, if necessary.

Cultivation can be performed under conditions suitable for cultivating the chosen bacterium in the method for producing a tripeptide γ-Glu-Val-Gly. For example, the cultivation can be performed under aerobic conditions for from <NUM> to <NUM> hours or for from <NUM> to <NUM> hours, the culture temperature during cultivation can be controlled within from <NUM> to <NUM> or within from <NUM> to <NUM>, and the pH can be adjusted between <NUM> and <NUM> or between <NUM> and <NUM>. The pH can be adjusted using an inorganic or organic acidic or alkaline substance such as urea, calcium carbonate or ammonia gas.

After cultivation, the tripeptide γ-Glu-Val-Gly can be collected from the culture medium. Specifically, the tripeptide γ-Glu-Val-Gly present outside of cells can be collected from the culture medium. Also, after cultivation, the tripeptide γ-Glu-Val-Gly can be collected from the bacterial cells, specifically, the cells can be disrupted, a supernatant can be obtained by removing solids such as the cells and the cell-disrupted suspension (so-called cell debris), and then the tripeptide γ-Glu-Val-Gly can be collected from the supernatant. Disruption of the cells can be performed using, for example, methods that are well-known in the art, such as ultrasonic lysis using high frequency sound waves, or the like. Removal of solids can be performed by, for example, centrifugation or membrane filtration. Collection of the tripeptide γ-Glu-Val-Gly from the culture medium or the supernatant etc can be performed using, for example, conventional techniques such as concentration, crystallization, ion-exchange chromatography, medium or high pressure liquid chromatography, or a combination of these.

The present invention will be more specifically explained with reference to the following examples.

coli strain MG1655 PL-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::KmR-Ptac4071ϕ10-gshBM165F(L029-<NUM>) was constructed as a model γ-EVG-producing strain. For that purpose, the strain MG1655 ilvG*MEDA ilvH** was used as a starting material. The strain MG1655 ilvG*MEDA ilvH** can be constructed from the E. coli strain K-<NUM> substr. MG1655 (ATCC <NUM>) using conventional recombinant methods and/or a genes chemical synthesis method. The strain MG1655 ilvG*MEDA ilvH** has a mutation in the ilvG gene (specifically, ilvG<NUM> mutation, which is the insertion of two base pairs (AA) at <NUM> position from the start of the gene, upstream of the sequence TGACTGGCA) that restores the frame-shift in the wild-type ilvG gene (specifically, the wild-type protein sequence. PLNQ& is replaced with. ), resulting in the restoration of acetohydroxyacid synthase II (AHAS II) activity (<CIT>; <CIT>), and also instills two mutations in ilvH gene that results in the replacement of <NUM>Ser with Phe and <NUM>Gly with Asp in the small subunit of acetolactate synthase III (AHAS III) (<CIT>). To overproduce valine in strain MG1655 ilvG*MEDA ilvH**, the native ilvG*MEDA operon was replaced with an artificial regulator region, which contains the phage lambda PL promoter linked to the modified Shine-Dalgarno sequence SD1 (SD sequence from pET22 plasmid), by the method developed by Datsenko and Wanner (<NPL>) called "λRed-dependent integration". According to this procedure, the PCR primers P1 (SEQ ID NO: <NUM>) and P2 (SEQ ID NO: <NUM>) were constructed. Oligonucleotide P1 (SEQ ID NO: <NUM>) is homologous to the region upstream of the ilvG gene and the region adjacent to the chloramphenicol resistance gene (cat), which was obtained from the chromosomal DNA of BW25113 cat-PL-yddG. Obtaining BW25113 cat-PL-yddG is described in detail (<CIT>, Russian patent <CIT>). BW25113 cat-PL-yddG strain was used as a template for PCR. Oligonucleotide P2 (SEQ ID NO: <NUM>) is homologous to both the ilvG region and the region downstream of the PL promoter, which was obtained from the template chromosome and contains the SD1 sequence. Conditions for PCR were as follows: denaturation for <NUM> at <NUM>; profile for two first cycles: <NUM> at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; profile for the last <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; final step: <NUM> at <NUM>. The resulting DNA fragment (<NUM> bp) (SEQ ID NO: <NUM>) was purified in an agarose gel and used for electroporation of the E. coli strain MG1655 containing the helper plasmid pKD46 with a temperature-sensitive replicon. The plasmid pKD46 (<NPL>) includes a <NUM>,<NUM> nt (<NUM>-<NUM>) DNA fragment from phage λ (GenBank, accession No. J02459) and contains the genes of the λRed homologous recombination system (gamma, beta, exo genes) under the control of the arabinose-inducible ParaB promoter. The plasmid pKD46 is necessary to integrate the DNA fragment into the bacterial chromosome.

Electrocompetent cells were prepared as follows: E. coli strain MG1655 was grown overnight at <NUM> in LB medium (<NPL>)) containing ampicillin (<NUM>/L), and the culture was diluted in <NUM> times with <NUM> of SOB medium (<NPL>)) with ampicillin (<NUM>/L) and L-arabinose (<NUM>). The cells were grown with aeration (<NUM> rpm) at <NUM> to an OD<NUM> of about <NUM> and then made electrocompetent by concentrating <NUM>-fold and washing three times with ice-cold deionized H<NUM>O. Electroporation was performed using <NUM>µl of cells and about <NUM> ng of DNA fragment (SEQ ID NO: <NUM>). Then, cells were incubated with <NUM> of SOC medium (<NPL>)) at <NUM> for <NUM>, placed onto plates containing LB-medium, agar (<NUM>%) and chloramphenicol (<NUM>µg/mL), and grown at <NUM> to select chloramphenicol resistant (CmR)-recombinants. Then, to eliminate the pKD46 plasmid, one passage on L-agar with Cm (<NUM>µg/mL) at <NUM> was performed, and the resulting individual colonies were tested for sensitivity to ampicillin. Thus, the strain MG1655 cat-PL-SD1-ilvG*MEDA was obtained.

The replacement of the native regulatory region of ilvG*MEDA operon with the PL-SD1 promoter marked with the chloramphenicol resistance gene was confirmed by PCR using locus-specific primers P3 (SEQ ID NO: <NUM>) and P4 (SEQ ID NO: <NUM>). Conditions for PCR verification were as follows: denaturation for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. DNA fragment, obtained in the reaction with the cells of MG1655 strain as a template, was <NUM> bp in length (SEQ ID NO: <NUM>). DNA fragment, obtained in the reaction with the cells of MG1655 cat-PL-SD1-ilvG*MEDA strain as a template, was <NUM> bp in length (SEQ ID NO: <NUM>). To eliminate CmR marker from the strain MG1655 cat-PL-SD1-ilvG*MEDA, cells were transformed with the plasmid pMW118-int-xis (ApR) (<CIT>). ApR clones were grown on LB-agar plates containing <NUM>/L ampicillin at <NUM>. Several tens of ApR clones were picked up and tested for chloramphenicol sensitivity. The plasmid pMW118-int-xis was eliminated from CmS cells by incubation on LB agar plates at <NUM>. Thus, the strain MG1655 PL-SD1-ilvG*MEDA was obtained.

γ-Glutamate-cysteine ligase (GshA) catalyzes the first step of γ-EVG biosynthesis. To increase expression of the gshA gene encoding GshA, the expression cassette ΔicdC::KmR-Ptac4071ϕ10-gshA50 (gshA50 means GshAL135F/Q144A, <CIT>) was introduced into strain MG1655 PL-SD1-ilvG*MEDA by P1-transduction (<NPL>)). Construction of the expression cassette ΔicdC::KmR-Ptac4071ϕ10-gshA50 is described in the Reference example <NUM>. Kanamycin-resistant transductants were selected and verified by PCR with the locus-specific primers P5 (SEQ ID NO: <NUM>) and P6 (SEQ ID NO: <NUM>). Conditions for PCR verification were as follows: denaturation step for <NUM> at <NUM>; profile for <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. There was no DNA fragment in the reaction with the cells of parental strain MG1655 PL-SD1-ilvG*MEDA as a template. The DNA fragment (SEQ ID NO: <NUM>) obtained in the reaction when using the MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 strain as a template, was <NUM> nt in length. The kanamycin resistance (KmR) marker was eliminated from MG1655 PL-SD1-ilvG*MEDA ΔicdC::KmR-Ptac4071ϕ10-gshA50 as described above. As a result, the strain MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 was obtained.

Glutathione synthetase (GshB) catalyzes the second step of γ-EVG biosynthesis. To overexpress glutathione synthetase, the cassette attB ϕ80::KmR-Ptac4071ϕ10-gshBM165F encoding mutant glutathione (y-EVG) synthetase with improved selectivity to γ-EV substrate (Reference example <NUM>) was introduced into strain MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 by P1-transduction. The donor strain MG1655 attB ϕ80::KmR-Ptac4071ϕ10-gshBM165F can be constructed as described in detail in Reference Example <NUM>. KmR transductants were selected and verified by means of PCR with locus-specific primers P7 (SEQ ID NO: <NUM>) and P8 (SEQ ID NO: <NUM>). Conditions for PCR verification were as follows: denaturation step for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. There was no DNA fragment in the reaction with the parental strain MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 as a template. DNA fragment (SEQ ID NO: <NUM>), obtained in the reaction with the cells of MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB phi80::KmR-Ptac4071ϕ10-gshB*M165F strain as a template, was <NUM> nt in length. As a result, the strain MG1655 PL-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::KmR-Ptac4071ϕ10-gshBM165F (L1029-<NUM>) was obtained.

To provide γ-EVG precursors, the regulator region of the kbl-tdh operon was modified, namely the native promoter region of the kbl-tdh operon was replaced with the PL promoter from phage λ, by the method of Red-dependent integration (<NPL>) described above. According to this procedure, the PCR primers P9 (SEQ ID NO: <NUM>) and P10 (SEQ ID NO: <NUM>) were constructed. Oligonucleotide P9 (SEQ ID NO: <NUM>) is homologous to the region upstream of the kbl gene and the region adjacent to the chloramphenicol resistance gene in the chromosomal DNA of the MG1655 cat-PL-SD1-ilvG*MEDA strain (Example <NUM>), which was used as a template for PCR. Oligonucleotide P10 (SEQ ID NO: <NUM>) is homologous to both the kbl region and the region downstream to the PL promoter in the chromosome of the MG1655 cat-PL-SD1-ilvG*MEDA strain. Conditions for PCR were as follows: denaturation for <NUM> at <NUM>; profile for two first cycles: <NUM> at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; profile for the last <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; final step: <NUM> at <NUM>. The obtained DNA fragment (<NUM> bp) (SEQ ID NO: <NUM>) was purified by "Silica Bead DNA Gel Extraction Kit" ("Thermo Scientific"), and used for electroporation of the E. coli strain MG1655 containing the plasmid pKD46. Chloramphenicol resistant recombinants were selected after electroporation. The replacement of the native regulatory region of kbl-tdh operon with the PL-SD1 promoter, marked with the Cm resistance gene, was confirmed by PCR using locus-specific primers P11 (SEQ ID NO: <NUM>) and P12 (SEQ ID NO: <NUM>). Conditions for PCR verification were as follows: denaturation for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. The DNA fragment obtained in the reaction with the MG1655 strain as a template was <NUM> nt in length (SEQ ID NO: <NUM>). The DNA fragment obtained in the reaction with the MG1655 cat-PL-kbl-tdh strain as a template was <NUM> nt in length (SEQ ID NO: <NUM>). Thus, the strain MG1655 cat-PL-SD1-kbl-tdh was obtained.

Then, the expression cassette cat-PL-SD1-kbl-tdh was introduced into the γ-EVG-producing strain MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-ghhA50 attB ϕ80::KmR-Ptac4071ϕ10-gshBM165F (L1029-<NUM>) by P1-transduction. As a result, the strain MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attBϕ80::KmR-Ptac4071ϕ10-gshBM165F cat-PL-SD1-kbl-tdh (L1031-<NUM>) was obtained. Evaluation of the strain L1031-<NUM> in comparison with the parent L1029-<NUM> demonstrated the positive effect of kbl-tdh operon overexpression on γ-EVG synthesis (Table <NUM>), namely, γ-EVG production increased more than <NUM> times and, as expected, the ratio of production of the byproduct γ-EV to production of γ-EVG was decreased.

To prevent glycine degradation due to a glycine decarboxylase reaction, the gcvP gene encoding a component of the glycine cleavage system was deleted by the method of Red-dependent integration described above. According to this procedure, the PCR primers P13 (SEQ ID NO: <NUM>) and P14 (SEQ ID NO: <NUM>) homologous to the both region adjacent to the gcvP gene and gene conferring kanamycin resistance in the template plasmid were constructed. The plasmid pMW118-(λattL-Km-λaattR) (<CIT>) was used as a template in PCR reaction. Conditions for PCR were as follows: denaturation step for <NUM> at <NUM>; profile for two first cycles: <NUM> at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; profile for the last <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; final step: <NUM> at <NUM>. The obtained DNA fragment (<NUM> bp) (SEQ ID NO: <NUM>) was purified by "Silica Bead DNA Gel Extraction Kit" ("Thermo Scientific") and used for electroporation of the E. coli strain MG1655, containing the plasmid pKD46. Kanamycin resistant recombinants were selected and the deletion of gcvP gene marked with KmR gene in selected mutants was verified by PCR using locus-specific primers P15 (SEQ ID NO: <NUM>) and P16 (SEQ ID NO: <NUM>). Conditions for PCR verification were as follows: denaturation step for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. The DNA fragment obtained in the reaction with the chromosomal DNA from parental gcvP+ strain MG1655 as a template was <NUM> nt in length (SEQ ID NO: <NUM>). The DNA fragment obtained in the reaction with the chromosomal DNA from mutant MG1655 ΔgcvP::KmR strain as a template was <NUM> nt in length (SEQ ID NO: <NUM>). As a result, the strain MG1655 ΔgcvP::KmR was obtained. The strain MG1655ΔgcvP::KmR was used as a donor for P1-mediated introduction of deletion of the gcvP gene into the strain MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attBϕ80::Ptac4071ϕ10-gshBM165F cat-PL-SD1-kbl-tdh, which was obtained from the strain L1031-<NUM> by the KmR marker excision. As a result, the strain MG1655 PL-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::Ptac4071ϕ10-gshBM165F cat-PL-SD1-kbl-tdh ΔgcvP::KmR (L1033-<NUM>) was selected. Evaluation of the strain L1033-<NUM> revealed the positive effect of gcvP gene disruption on γ-EVG production; and as expected, the byproduct γ-EV was decreased (Table <NUM>).

To demonstrate the effect of ilvGMEDA operon expression on γ-EVG production, the expression cassette PL-SD1-ilvG*MEDA in the strain MG1655 PL-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::Ptac4071ϕ10-gshBM165F cat-PL-SD1-kbl-tdh ΔgcvP::Km (L1033-<NUM>) was replaced with the wild-type ilvGMEDA operon. To this end, at first, the strain MG1655 PL-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::Ptac4071ϕ10-gshBM165F PL-SD1-kbl-tdh ΔgcvP::KmR (L1034-<NUM>) was obtained from the strain L1033-<NUM> by the CmR marker elimination. Then, the expression cassette PL-SD1-ilvG*MEDA in the strain L1034-<NUM> was replaced with the cassette PL-SD1-ilvG*M-ΔilvE::cat-DA by means of P1-transduction. The strain MG1655 PL-SD1-ilvG*M-ΔilvE::cat-DA that was used as a donor strain can be constructed as described in detail in Reference example <NUM>. Thus, the strain L1034-<NUM> PL-SD1-ilvG*M-ΔilvE::cat-DA that requires isoleucine and valine for growth in minimal medium was obtained.

Then, the expression cassette PL-SD1-ilvG*M-ΔilvE::cat-DA in the chromosome of the strain L1034-<NUM> PL-SD1-ilvG*M-ΔilvE::cat-DA was replaced with the wild-type ilvGMEDA operon by P1-transduction from E. coli strain MG1655; prototrophic transductants were selected in M9 minimal medium. Restoration of the wild-type ilvGMEDA operon in the resulting strain MG1655 ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::Ptac4071ϕ10-gshBM165F PL-SD1-kbl-tdh ΔgcvP::KmR (L1040-<NUM>) was confirmed by PCR with locus-specific primers P3 (SEQ ID NO: <NUM>) and P4 (SEQ ID NO: <NUM>). Conditions for PCR verification were as follows: denaturation step for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. DNA fragment, obtained in the reaction with the cells of the parental strain as a template, was <NUM> nt in length (SEQ ID NO: <NUM>). The DNA fragment obtained in the reaction with the cells of L1040-<NUM> strain as a template was <NUM> nt in length (SEQ ID NO: <NUM>). As seen in Table <NUM>, the substitution of the efficient PL-SD1-ilvGMEDA expression cassette with the wild-type ilvGMEDA operon into the chromosome of L1040-<NUM> strain resulted in significantly decreasing γ-EVG production as compared to L1034-<NUM>.

The decrease in <NUM>-ketoglutarate dehydrogenase (KGDH) activity was supposed to be useful for γ-EVG production due to its positive influence on Glu synthesis. The down-regulation of the sucAB genes that encode two subunits of KGDH was previously achieved by the replacement of the native promoter with the artificial Ptac-derived promoter, Ptac21 (<CIT>). It has been demonstrated in E. coli that the expression cassette cat-Ptac21-sucAB decreased KGDH activity by <NUM> %, thereby elevating Glu accumulation <NUM>-fold (<CIT>). This cassette was also introduced into the γ-EVG-producing strain MG1655 PL-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::Ptac4071ϕ10-gshBM1651 PL-SD1-kbl-tdh ΔgcvP::KmR (L1034-<NUM>) by P1 transduction. However, no positive effect on γ-EVG or γ-EV accumulation was observed in fed-batch EVG-cultivation. However, when using a flask for cultivation, the positive effect of sucAB genes down-regulation was observed (Table <NUM>), namely, γ-EVG production increased about <NUM> times. Evidently, the difference in aeration conditions in flasks and jars was a key reason for the difference in evaluation results.

Experimental procedure: cells from stock tube (stored in <NUM> % glycerol, <NUM> % NaCl at -<NUM>) were placed on L-agar (yeast extract (Dia-M) <NUM>/L, peptone (Dia-M) <NUM>/L, NaCl <NUM>/L, agar <NUM>/L). Cells from about one half of the plate surface were inoculated into <NUM> MS(+pyr) medium (Table <NUM>) and cultivated for <NUM> hour at <NUM> at rotary shaker. After that, additional glucose was added to a final concentration of <NUM>/L. After this addition, strains were cultivated for <NUM> hours at <NUM> on a rotary shaker. The total cultivation time was <NUM>.

To improve γ-EVG export, the expression of the tolC gene, that encodes an outer membrane protein involved in a range of tripartite efflux complexes (Benz R. , <NUM>), was increased. To this end, the expression cassette cat-PLtac-tolC (Reference example <NUM>) was introduced into the γ-EVG-producing strain MG1655 PL-SD1-ilvG*MEDA ΔicdC::Ptac4071ϕ10-gshA50 attB ϕ80::Ptac4071ϕ10-gshBM165F PL-SD1-kbl-tdh ΔgcvP::KmR (L1034-<NUM>) by P1-transduction. Evaluation of the obtained strain L1034-<NUM> cat-PLtac-tolC (L1036-<NUM>) in comparison with the parental strain demonstrated a positive effect of tolC gene overexpression on γ-EVG production, namely more than a <NUM>% increase in γ-EVG accumulation when tolC is overexpressed (Table <NUM>).

A DNA fragment containing the gshBM165F gene was re-cloned from the plasmid pUC19-EcGshB*M165F (Reference example <NUM>) into the integrative vector pAH162-λattL-TcR-λattR (<NPL>) using the PstIlSacI restriction sites, yielding the delivery plasmid pAH162-GshB*M165F. The gene gshBM165F was inserted into the native ϕ80 locus by ϕ80 integrase-mediated recombination (Minaeva N. Then, to achieve a high expression level, the regulatory region Ptac4071ϕ10 was introduced upstream of the gshBM165F gene by the Red-dependent integration method described above. According to this procedure, the PCR primers P17 (SEQ ID NO: <NUM>) and P18 (SEQ ID NO: <NUM>) were constructed. Oligonucleotide P17 (SEQ ID NO: <NUM>) is homologous to the region upstream of the gshBM165F gene and the region adjacent to the KmR gene in the chromosomal DNA of the MG1655 PL-SD1-kbl-tdh ΔgcvP PL-SD1-ilvG*MEDA ΔicdC::KmR-Ptac4071ϕ10-gshA50 strain, which was used as a template for PCR. Oligonucleotide P18 (SEQ ID NO: <NUM>) is homologous to both the gshBM165F region and the region downstream to the Ptac4071ϕ10 regulatory region in the chromosome of the MG1655 PL-SD1-kbl-tdh ΔgcvP PL-SD1-ilvG*MEDA ΔicdC::KmR-Ptac4071ϕ10-gshA50 strain. Conditions for PCR were as follows: denaturation for <NUM> at <NUM>; profile for two first cycles: <NUM> at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; profile for the last <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; final step: <NUM> at <NUM>. The resulting <NUM> bp DNA fragment (SEQ ID NO: <NUM>) was purified by "Silica Bead DNA Gel Extraction Kit" ("Thermo Scientific") and used for electroporation of the strain MG1655 attB phi80::gshBM165F containing the plasmid pKD46. Kanamycin-resistant recombinants were selected; the introduction of Ptac4071ϕ10 promoter marked with the KmR resistance gene was confirmed by PCR using locus-specific primers P7 (SEQ ID NO: <NUM>) and P8 (SEQ ID NO: <NUM>). Conditions for PCR verification were as follows: denaturation for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. The DNA fragment obtained in the reaction with the MG1655 strain used as a template was <NUM> nt in length (SEQ ID NO: <NUM>). The DNA fragment obtained in the reaction with the MG1655 attB phi80::KmR-Ptac4071cp10-gshB*M165F strain as a template was <NUM> nt in length (SEQ ID NO: <NUM>). As a result, the strain MG1655 attB phi80::KmR-Ptac4071ϕ10-gshBM165F was obtained.

The cassette PL-SD1-ilvG*M-ΔilvE::cat-DA was constructed by the method of Red-dependent integration described above. According to this procedure, the PCR primers P19 (SEQ ID NO: <NUM>) and P20 (SEQ ID NO: <NUM>) were constructed. These primers are homologous to both the region adjacent to the ilvE gene and the gene that confers chloramphenicol resistance in the template plasmid pMW-attL-Cm-attR (<CIT>). Conditions for PCR were as follows: denaturation step for <NUM> at <NUM>; profile for two first cycles: <NUM> at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; profile for the last <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; final step: <NUM> at <NUM>. The obtained DNA fragment (<NUM> bp) (SEQ ID NO: <NUM>) was purified by "Silica Bead DNA Gel Extraction Kit" ("Thermo Scientific") and used for electroporation of the strain MG1655 PL-SD1-ilvG*MEDA containing the plasmid pKD46. Chloramphenicol resistant recombinants were selected and the deletion of ilvE gene marked with CmR gene in selected mutants was verified by PCR using locus-specific primers P21 (SEQ ID NO: <NUM>) and P22 (SEQ ID NO: <NUM>). Conditions for PCR verification were following: denaturation step for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. The DNA fragment obtained in the reaction with the parental strain MG1655 PL-SD1-ilvG*MEDA as a template was <NUM> nt in length (SEQ ID NO: <NUM>). The DNA fragment obtained in the reaction with the MG1655 PL-SD1-ilvG*M-ΔilvE::cat-DA strain as a template, was <NUM> nt in length (SEQ ID NO: <NUM>).

Using pSF12-gshA*<NUM> (described in <CIT>) as a template, PCR was carried out with primer pair P23 (SEQ ID NO: <NUM>) and P24 (SEQ ID NO: <NUM>) to add scaffold nucleotides at the edge of the gshA * <NUM> fragment for fusion-PCR with a chemically synthesized Ptac4071f10 fragment (SEQ ID NO: <NUM>). Then, the gshA*<NUM> fragment with scaffold and Ptac4071f10 fragment were mixed and used as the template in fusion-PCR using primer pair P24 (SEQ ID NO: <NUM>) and P25 (SEQ ID NO: <NUM>) to construct Ptac4071f10-gshA*<NUM> fragment. The Ptac4071f10-gshA*<NUM> fragment was cloned into XbaI restriction site of pMW <NUM>-attL-kan-attR plasmid (<CIT>, <CIT>) using in-fusion technique (In-Fusion HD cloning Kit, Clontech). The constructed plasmid was designated as pMW118-attL-kan-attR-Ptac4071f10-gshA*<NUM>. KmR-Ptac4071f10-gshA50 cassette was amplified by PCR using primer pair P26 (SEQ ID NO: <NUM>) and P27 (SEQ ID NO: <NUM>), and pMW118-attL-kan-attR-Ptac4071f10-gshA*<NUM> as a template. The obtained DNA fragment was introduced into strain E. coli K-<NUM> MG1655 strain (ATCC <NUM>) using λ-red technique (<NPL>; <NPL>). This constructed strain was designated as MG1655ΔicdC::attL-kan-attR-Ptac4071f10-gshA*<NUM> harboring the cassette ΔicdC::KmR-Ptac4071ϕ10-gshA50.

At first, gshB expression plasmid pUC19-Plac-gshB was prepared. A gshB fragment was amplified using primer pair P28 (SEQ ID NO: <NUM>) and P29 (SEQ ID NO: <NUM>), and chromosomal DNA of MG1655 strain as a template. The gshB fragment was cloned at XbaI site of pUC19 (New England Biolabs) using in-fusion technique (In-Fusion HD cloning Kit, Clontech). Then, pUC19-Plac-gshB*M165F was prepared by carrying out PCR using primer pairs P30 (SEQ ID NO: <NUM>) and P31 (SEQ ID NO: <NUM>), and pUC19-Plac-gshB as a template. After digesting the obtained PCR fragment with DpnI to degrade the PCR template pUC19-Plac-gshB, competent cells JM109 (available from Takara Bio) were transformed with the PCR solution to obtain gshB*M165F expression plasmid pUC19-Plac-gshB*M165F.

Using pUC19-Plac-gshB*M165F as a template, a PCR was carried out using primer pairs P32 (SEQ ID NO: <NUM>) and P33 (SEQ ID NO: <NUM>) to add scaffold nucleotides at the edge of gshB*M165F fragment for fusion-PCR with chemically synthesized Ptac4071f10 fragment (SEQ ID NO: <NUM>). Then, gshB*M165F fragment with scaffold and Ptac4071f10 fragment were mixed and used as template in fusion-PCR using primer pairs P25 (SEQ ID NO: <NUM>) and P33 (SEQ ID NO: <NUM>) to construct Ptac4071f10-gshB*M165F fragment. The Ptac4071f10-gshB*M165F fragment was cloned into XbaI site of pMW <NUM>-attL-cat-attR plasmid by in-fusion technique (In-Fusion HD cloning Kit, Clontech). This constructed plasmid was designated as pMW118-attL-cat-attR-Ptac4071f10-gshB*M165F. CatR-Ptac4071f10-gshB*M165F cassette was amplified by PCR using primer pair P34 (SEQ ID NO: <NUM>) and P35 (SEQ ID NO: <NUM>), and pMW118-attL-cat-attR-Ptac4071f10-gshB*M165F as a template. The obtained DNA fragment was introduced into strain MG1655ΔicdC::attL-kan-attR-Ptac4071f10-gshA*<NUM> (Reference example <NUM>) by λ-red technique. Constructed strain was designated as MG1655ΔicdC::attL-kan-attR-Ptac4071f10-gshA*<NUM>ΔgshB::attL-cat-attR-Ptac4071f10-gshB*M165F harboring the cassette attB ϕ80::CatR-Ptac4071ϕ10-gshBM165F.

The tolC gene was overexpressed using the method of Red-dependent integration (<NPL>). According to this procedure, the PCR primers P36 (SEQ ID NO: <NUM>) and P37 (SEQ ID NO: <NUM>), which are homologous to both regions adjacent to the tolC gene and regions adjacent to the chloramphenicol resistance gene (CmR) and the PLtac promoter in the template chromosome, were constructed. The chromosome of the strain MG1655 cat-PL-tac-xylE, which contains hybrid PLtac promoter, was used as the template in PCR reaction. The strain MG1655 cat-PLtac-xylE can be constructed as described in detail in <CIT>. Conditions for PCR were as follows: denaturation for <NUM> at <NUM>; profile for two first cycles: <NUM> at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; profile for the last <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> sec at <NUM>; final step: <NUM> at <NUM>. The obtained DNA fragment (<NUM> bp) (SEQ ID NO: <NUM>) was purified by "Silica Bead DNA Gel Extraction Kit" ("Thermo Scientific"), and used for electroporation of the E. coli strain MG1655 containing the plasmid pKD46. Chloramphenicol resistant recombinants were selected after electroporation. The replacement of the native regulatory region of tolC gene with the PLtac promoter region, marked with the Cm resistance gene, was confirmed by PCR using locus-specific primers P38 (SEQ ID NO: <NUM>) and P39 (SEQ ID NO: <NUM>). Conditions for PCR verification were the following: denaturation for <NUM> at <NUM>; profile for the <NUM> cycles: <NUM> sec at <NUM>, <NUM> sec at <NUM>, <NUM> at <NUM>; final step: <NUM> at <NUM>. DNA fragment (SEQ ID NO: <NUM>), obtained in the reaction with the cells of MG1655 strain used as a template, was <NUM> nt in length. DNA fragment (SEQ ID NO: <NUM>), obtained in the reaction with the cells of MG1655 cat-PLtac-tolC strain as a template, was <NUM> nt in length. Then, the plasmid pKD46 was eliminated by cultivation at <NUM>. Thus, the E. coli strain MG1655 harboring the expression cassette cat-PLtac-tolC was constructed.

For each of the above examples, the following culture conditions were used for EVG-cultivation.

coli strains were maintained on LB agar plates at <NUM>. LB agar (<NUM>%) was used for pre-cultivation of all investigated strains at <NUM> overnight during <NUM>.

Seed culture was conducted in GALLENCAMP shaker in <NUM>-mL Erlenmeyer flask on LB liquid media (volume of the media was <NUM>) during <NUM>. Temperature of cultivation was <NUM>, rotation speed was <NUM> rpm. One loop of biomass from pre-cultivated plate was used for inoculation of seed culture.

Main culture was conducted in <NUM> S-Jars (ABLE Biott). Cultivating conditions were as follows: agitation at <NUM> rpm was fixed, aeration <NUM>/<NUM> vvm, temperature <NUM>, pH <NUM> was maintained using NH<NUM> gas. Dissolved Oxygen level was controlled as follows: if DO level become <NUM>% (DO limiting condition occurred) DO level was maintained at <NUM>% after DO limitation stopped by dropping down agitation. If DO level did not reach <NUM>% no DO control was used. Media volume was <NUM>, inoculation volume of seed culture was <NUM>%.

The composition of the medium used for main culture is shown in Table <NUM>.

After <NUM> of cultivation, glucose feeding solution (concentration <NUM>/L) was continuously added to maintain glucose level in the culture broth within the range of <NUM>-<NUM>/L. Culture time of main process in total was <NUM>.

Similarly, for each of the above examples, the HPLC analysis was conducted as follows. Analysis of γ -EVG was done using high performance liquid chromatography (HPLC). A modified AccQ*Tag method was used for the analysis of samples, with pre-column derivatization.

Claim 1:
A method for producing γ-Glu-Val-Gly comprising:
(i) cultivating a γ-Glu-Val-Gly-producing bacterium belonging to the family Enterobacteriaceae in a culture medium so that the γ-Glu-Val-Gly is produced and accumulates in the culture medium or the cells of the bacterium, or both, and
(ii) collecting the γ-Glu-Val-Gly from the culture medium or the cells of the bacterium, or both,
wherein the bacterium has been modified to overexpress a gene encoding a protein having L-threonine <NUM>-dehydrogenase activity and a gene encoding a protein having <NUM>-amino-<NUM>-oxobutanoate coenzyme A ligase activity.